Comprehensive control method for quality of compound Pien Tze Huang buccal tablets

Abstract

The invention belongs to the field of medicine detection, and particularly discloses a comprehensive control method for the quality of compound Pien Tze Huang lozenges, which comprises the following steps of: fishing targeted protein active ingredients of the compound Pien Tze Huang lozenges by respectively adopting a surface plasma resonance instrument with a built-in TNFR2 protein chip, a built-in TLR4 protein chip or a built-in TLR5 protein chip; the biological effect of the compound Pien Tze Huang lozenge is detected by adopting an SPR method based on the fished anti-inflammatory active ingredients, and the fingerprint spectrum of the compound Pien Tze Huang lozenge and the content of the ingredients in the fingerprint spectrum are detected by adopting an UPLC method. The surface plasmon resonance method and the UPLC method are combined to control the quality of the compound Pien Tze Huang lozenge, evaluation of the overall drug effect and accurate determination of component composition are combined, the quality of the compound Pien Tze Huang lozenge is controlled more comprehensively, and the method has practical application value.

Description

Technical Field

[0001] The invention belongs to the field of drug detection, and particularly relates to a comprehensive control method for the quality of compound Pien Tze Huang lozenges. Background Art

[0002] Compound Pien Tze Huang lozenges are a traditional Chinese medicine developed and manufactured by Zhangzhou Pien Tze Huang Pharmaceutical Company (National Medicine Approval Number B20050066). The formula consists of six medicinal ingredients: Wedelia chrysanthemum, Sarcandra chinensis, Scrophularia ningpoensis, Ophiopogon japonicus, Licorice, and Pien Tze Huang, supplemented with menthol. It can clear away heat and detoxify, relieve sore throat and pain, promote salivation and relieve pain. It is used for acute and chronic pharyngitis caused by wind-heat and excessive heat in the spleen and stomach.

[0003] As a modern formulation of a traditional Chinese medicine compound, Compound Pien Tze Huang lozenges have a complex chemical composition, limited research on their material basis and mechanism of action, and low quality control. Existing studies have used reversed-phase HPLC to determine the content of isofraquinone in Compound Pien Tze Huang lozenges, TLC to identify Wedelia chrysanthemi and Glycyrrhiza uralensis, and TLC to identify isofraquinone with Glycyrrhiza uralensis and Radix Ophiopogonis. These analytical methods rely solely on single-index content determination and thin-layer colorimetric identification, and are unable to systematically evaluate the quality of Compound Pien Tze Huang lozenges. Modern scientific and technological methods are urgently needed for effective analysis. Academician Liu Changxiao and others proposed Chinese medicine quality markers (Q-markers) as a new strategy to strengthen the quality control of Chinese medicine compound prescriptions. Q-markers include not only Chinese medicine quality chemical markers (Q-chemomarkers) but also Chinese medicine quality biomarkers (Q-biomarkers). Therefore, establishing a quality control method that integrates chemical quality markers and biological quality markers has important practical significance for improving the quality standards of compound Pien Tze Huang lozenges.

[0004] Surface plasmon resonance (SPR) is a biosensor technology based on photonics and electromagnetics, used to study interactions between biomolecules. Its high sensitivity, real-time monitoring, and high specificity make it a crucial tool for detecting the biological effects of traditional Chinese medicine (TCM) preparations. It not only helps identify and verify the active ingredients of TCMs but also provides strong support for research into their mechanisms of action. While SPR technology holds enormous potential for detecting TCM preparations, its practical application still faces several difficulties and challenges. A major challenge is its limited ability to handle the complexity of TCM extracts. Effectively screening for potentially pharmacologically active components from complex TCM extracts presents a significant challenge.

[0005] Traditional Chinese medicine fingerprints are spectra or chromatograms of the chemical components of traditional Chinese medicines obtained with the help of spectral or chromatographic techniques. They have the characteristics of holistic, macroscopic, and fuzzy analysis. They can achieve the purpose of overall quality control by describing the overall characteristics of traditional Chinese medicines. They are an important means of detecting chemical quality markers of traditional Chinese medicines. An ideal fingerprint spectrum can not only achieve the purpose of qualitative identification, but also achieve baseline separation of some drug spectra to realize quantitative analysis, satisfying qualitative identification while controlling the absolute content of representative components. This can greatly improve the quality control level of traditional Chinese medicines and their compound preparations. However, the method of combining traditional Chinese medicine fingerprints with multi-component content determination in the application of compound traditional Chinese medicines still faces technical difficulties in the selection of test sample preparation methods, the exploration of liquid chromatography conditions, the establishment of common fingerprint patterns, and the selection and identification of chromatographic peaks.

[0006] So far, there has been no report or application of the comprehensive quality control of Compound Pien Tze Huang lozenges using SPR technology and fingerprint or fingerprint combined with multi-component content determination. Summary of the Invention

[0007] The purpose of the present invention is to establish a comprehensive control method for the quality of compound Pien Tze Huang lozenges, so as to comprehensively and accurately control the quality of compound Pien Tze Huang lozenges and ensure the efficacy of compound Pien Tze Huang lozenges.

[0008] The present invention provides a quality control method for compound Pien Tze Huang buccal tablets, which is characterized by: using a surface plasmon resonance method to carry out targeted protein active ingredient fishing on the compound Pien Tze Huang buccal tablets; the protein is TNFR2 protein, TLR4 protein and / or TLR5 protein;

[0009] When the protein is TNFR2 protein, the active ingredients fished out are isofraquinone, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apigenin isoliquiritin, isoliquiritin, notoginsenoside R1, ginsenoside Rh1, ginsenoside Rb1 and ginsenoside Rd;

[0010] When the protein is TLR4 protein, the activities fished out are isofraquinone, neoliquiritin, nepetaside, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiol isoliquiritin, liquiritigenin and formononetin;

[0011] When the protein is TLR5 protein, the activities detected are neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid.

[0012] Furthermore, the fishing comprises the following steps:

[0013] A1. Constructing a protein chip: Dissolve TNFR2 protein, TLR4 protein or TLR5 protein in water, then dilute with sodium acetate buffer to a 30-50 μg / ml protein solution. Take the protein solution and couple it with the sensor chip.

[0014] A2 Preparation of fishing test solution: Take compound Pien Tze Huang lozenges, add 60% methanol solution to extract, centrifuge the extract, filter the supernatant, dry the filtrate, add DMSO to re-dissolve, and then add 1× PBS solution to mix.

[0015] 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;

[0016] Fishing conditions were as follows: mobile phase: PBS buffer solution containing 5% DMSO; sample flow rate: 5 μL min -1 Injection time: 60 s; Binding time: 180 s; Dissociation time: 60 s; Dissociation reagent: 0.5% TFA solution, Dissociation reagent volume: 2 μL, Dissociation reagent incubation time: 20 s; Recovery reagent: 50 mM NH4HCO3 solution, Recovery reagent volume: 10 μL; Number of cycles: 10 times;

[0017] A4 uses UPLC-Q-TOF-MS method to perform qualitative detection on the fishing component recovery liquid obtained in step A3.

[0018] Furthermore, the concentration of the sodium acetate buffer in step A1 is 10 mM, and the pH value is 4-4.5; the protein solution is 50 μg / ml; the coupling is amino coupling; the parameters of the amino coupling are as follows: sensor chip model: CM7 or CM5, coupling reagents: EDC and NHS, protein solution flow rate: 5 μl / min, protein contact time: 720 s, temperature: 25°C, blocking reagent: ethanolamine, and blocking time: 7 min;

[0019] And / or: the mass volume ratio of the compound Pien Tze Huang lozenges in step A2 to the 60% methanol solution is 0.1 g: 5-25 ml; the extraction is ultrasonic extraction, the time is 20-60 min; the centrifugation speed is 5000-25000 r, the time is 5-25 min; the volume ratio of the filtrate, DMSO and PBS 1× solution is 0.5-1.5 mL: 100 μL: 1900 μL.

[0020] Furthermore, when the protein solution in step A1 is a TNFR2 protein solution or a TLR4 protein solution, the pH value of the sodium acetate buffer is 4; when the protein solution is a TLR5 protein solution, the pH value of the sodium acetate buffer is 4.5; the sensor chip model is CM5, and the volume ratio of the protein solution, EDC and NHS, and ethanolamine is 100:200:140; the volume ratio of the EDC and NHS is 1:1.

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

[0022] ① Preparation of reference solution: Take isoflurane, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiose isoliquiritin, isoliquiritin, notoginsenoside R1, ginsenoside Rh1, ginsenoside Rb1 and ginsenoside Rd reference substances, add methanol to dissolve as reference solution for qualitative targeting of TNFR2 protein active ingredients;

[0023] And / or: take isoflurane, neoliquiritin, nepetaside, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiosyl isoliquiritin, liquiritigenin and formononetin reference substances, add methanol to dissolve as reference substance solution for qualitatively targeting TLR4 protein active ingredients;

[0024] And / or: Take neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid reference substances and dissolve them in methanol as reference solution for qualitatively targeting the active ingredient of TLR5 protein;

[0025] ② Detection: The recovered liquid of fishing ingredients was dried and dissolved in 50% methanol. The dissolved liquid and the reference solution obtained in step ① were injected into UPLC-Q-TOF-MS for detection respectively;

[0026] Chromatographic conditions were as follows: chromatographic column: C18 column; mobile phase: acetonitrile-0.1% formic acid water; gradient elution program: 0-0.5 min, 5% acetonitrile; 0.5-5 min, 5% acetonitrile; 5-13 min, 5-13% acetonitrile; 13-17 min, 13% acetonitrile; 17-29 min, 13-25% acetonitrile; 29-31 min, 25% acetonitrile; 31-36 min, 25-3 0% acetonitrile; 36-41 min, 30% acetonitrile; 41-48 min, 30-35% acetonitrile; 48-52 min, 35% acetonitrile; 52-53 min, 35-38% acetonitrile; 53-56 min, 38-48% acetonitrile; 56-66 min, 48-57% acetonitrile; 66-72 min, 57-70% acetonitrile; 72-78 min, 70-85% acetonitrile;

[0027] The mass spectrometry conditions were as follows: ion source: electrospray ion source, negative ion mode, data acquisition mode: MSe full scan, range m / z 50-1500.

[0028] Furthermore, the mass concentration of each reference substance in the reference solution of step ① is 0.5-1.5 μg·mL -1

[0029] And / or: the C18 chromatographic column in the chromatographic conditions of step ② is Waters CORTECS UPLC C 18 Chromatographic column, 2.1 mm × 100 mm, 1.6 μm, flow rate 0.25 mL min -1, column temperature 45°C; injection volume 2 μL;

[0030] The mass spectrometry conditions were: capillary voltage: -3.0 kV, desolvation gas flow: nitrogen, flow rate: 800 L·h -1 , desolvation temperature: 500℃, cone gas flow: nitrogen, cone voltage: 30.0V, flow rate: 50L·h -1 , collision gas: argon, collision energy: 10-55eV, ion source temperature: 120℃.

[0031] The present invention also provides a method for detecting the biological effect of the compound Pien Tze Huang lozenges, which adopts surface plasmon resonance detection and comprises the following steps:

[0032] ① Using a surface plasmon resonance instrument to measure the RU value of the compound Pien Tze Huang lozenges and the RU value of isochlorogenic acid B, wherein the protein chip in the surface plasmon resonance instrument is a TNFR2 protein chip;

[0033] ② The ratio is calculated using the following formula. If the ratio is not less than 1, the biological effect test of Compound Pien Tze Huang Lozenges is qualified;

[0034] Ratio = RU value of Compound Pien Tze Huang lozenges / RU value of isochlorogenic acid B.

[0035] Furthermore, the steps for determining the RU value of the compound Pien Tze Huang lozenges and the RU value of isochlorogenic acid B are as follows:

[0036] The compound Pien Tze Huang lozenge solution and the isochlorogenic acid B reference solution were placed in a surface plasmon resonance test plate loaded with a TNFR2 protein chip. SPR assay conditions were: association time: 60 s; flow rate: 30 μl / min, dissociation time: 60 s; temperature: 25°C; running buffer: 5% DMSO in PBS.

[0037] The compound Pien Tze Huang buccal tablet test solution is prepared by taking the compound Pien Tze Huang buccal tablet and preparing the fishing test solution according to the method described in step A2;

[0038] The isochlorogenic acid B reference solution is prepared by dissolving isochlorogenic acid B in DMSO, adding PBS solution to the dissolving solution and mixing the solution with a PBS solution containing DMSO; the concentration of isochlorogenic acid B in the dissolving solution is 10 mM; the volume ratio of the dissolving solution, PBS solution and PBS solution containing DMSO is 5 μl:95 μl:900 μl;

[0039] The TNFR2 protein chip is prepared by taking TNFR2 protein according to the method for constructing a protein chip described in step A1.

[0040] The present invention also provides a UPLC detection method for active ingredients in compound Pien Tze Huang lozenges that target TNFR2 protein, TLR4 protein, and / or TLR5 protein. The method uses UPLC to detect the fingerprint of compound Pien Tze Huang lozenges and determines the content of the ingredients in the fingerprint. The method specifically comprises the following steps:

[0041] 1) Preparation of reference solution: Take the reference substance and dissolve it in methanol solution;

[0042] 2) Preparation of test solution: Take the compound Pien Tze Huang buccal tablets to be tested, extract with methanol solution, filter, and obtain the filtrate;

[0043] 3) Separately pipette the reference solution and the test solution into a high performance liquid chromatograph; the chromatographic conditions are as follows:

[0044] Chromatographic column: octadecylsilane bonded silica as filler; mobile phase: formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B; gradient elution program: 0-3 min, 5% B→13% B; 13-16 min, 13% B→15.5% B; 16-25 min, 15.5% B→21% B; 25-28 min, 21% B; 28-32 min, 21% B→22% B; 32-41 min, 22% B→30% B.

[0045] Furthermore, the concentration of the methanol solution in step 1) is 50% to 100%; the concentration of each reference substance in the reference solution is 1 to 50 μg·mL -1 ;

[0046] The reference substances are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid, isoflurane, liquiritin, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoside C and / or isoliquiritin.

[0047] And / or: in step 2), the mass volume ratio of the compound Pien Tze Huang lozenge to be tested and the methanol solution is 1-5 g:10-100 ml; the concentration of the methanol solution is 50%-100%, v / v.

[0048] And / or: Step 3) The chromatographic conditions are: Chromatographic column: Welch UltimateAQ-C 18 Chromatographic column, 2.1 mm × 150 mm, 1.8 μm, column temperature 30-35 °C, injection volume 2 μL, flow rate 0.25 mg min -1 , detection wavelength 330nm.

[0049] Furthermore, the fingerprint of the compound Pien Tze Huang lozenge to be tested should present 18 characteristic peaks, of which 13 peaks should correspond to the retention times of the peaks of the corresponding reference substance; the fingerprint is preferablyFigure 7 A.

[0050] Furthermore, the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid, isoflurane, liquiritin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoside C and / or isoliquiritin in the compound Pien Tze Huang lozenges to be tested are calculated using peak area according to the external standard method.

[0051] In the early stage of the present invention, network pharmacology analysis revealed that the compound Pien Tze Huang lozenges mainly exerted their anti-inflammatory effects by regulating the TLP channel and the TNF signaling pathway. Therefore, surface plasmon resonance (SPR) was used to fish for the active ingredients in the compound Pien Tze Huang lozenges that targeted the corresponding proteins using a protein fishing chip constructed with TNFR2 protein, TLR4 protein or TLR5 protein. A total of 10 components were fished out from the TNFR2 protein, namely, isofraquinone, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiosyl isoliquiritin, isoliquiritin, notoginsenoside R1, ginsenoside Rh1, ginsenoside Rb1 and ginsenoside Rd. The KD values were between 1.06 and 8.85 μM, and the molecular docking energy range of the compounds binding to the TNFR2 target was -7.85 to -4.49 kcal·mol -1 A total of 10 components were fished out from the TLR4 protein, namely, isofraquinone, neoliquiritin, nepetaside, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiol isoliquiritin, liquiritigenin and formononetin, with KD values ranging from 0.78 to 18.70 μM. The molecular docking energy range of the compounds and TLR4 target binding was -5.50 to -4.58 kcal·mol -1 Three components were identified from the TLR5 protein, namely neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid. Their KD values were 57.24 μM for neochlorogenic acid, 9.69 μM for chlorogenic acid, and 38.53 μM for cryptochlorogenic acid, respectively. The molecular docking energy range was -6.15 to -5.86 kcal·mol -1 The molecular docking binding can verify the affinity, laying the foundation for the biological effect evaluation of Compound Pien Tze Huang Lozenges.

[0052] Using surface plasmon resonance (SPR) technology, a protein fishing chip constructed with TNFR2 protein was used to assess the biological effects of Compound Pien Tze Huang lozenges, using isochlorogenic acid B as a positive control. Methodological validation revealed that the positive control, isochlorogenic acid B, had a precision RSD of 0.6%, sample precision RSD of 1.4%, repeatability RSD of 4.3%, and stability RSD of 4.1%, demonstrating excellent precision, repeatability, and stability. RU values for all batches of samples ranged from 118.4 to 403.3, while RU values for isochlorogenic acid B ranged from 27.8 to 30.5, respectively. The ratios ranged from 4.2 to 13.2, all exceeding 1. This demonstrates that the constructed biological effect assay is effective in evaluating the quality of Compound Pien Tze Huang lozenges.

[0053] The active ingredients of Compound Pien Tze Huang buccal tablets were screened using the SPR method for TNFR2 protein, TLR4 protein, and TLR5 protein targets. These active ingredients revealed that Compound Pien Tze Huang buccal tablets can exert anti-inflammatory effects through TNFR2, TLR4, and TLR5 protein targets, and their contents need to be effectively detected. The present invention uses the UPLC method to establish a UPLC fingerprint of Compound Pien Tze Huang buccal tablets, and the content of these anti-inflammatory ingredients is determined by the fingerprint. The UPLC method established in the present invention has precision, repeatability and stability, with relative retention time RSDs of ≤0.51% and relative peak area RSDs of ≤3.34%. 13 common peaks are identified, and the similarities between the fingerprints of samples of each batch and the control are greater than 0.900, indicating good similarity. The method can quantify 12 components in the compound Pien Tze Huang lozenges, and the 12 components show a good linear relationship within the linear range (r≥0.9989). The RSDs of the precision, repeatability and stability are all ≤2.95%, and the average sample recovery rate is 99.09% to 101.85% (RSD≤2.82%), which meets the requirements of quantitative analysis and lays a foundation for improving the chemical quality standards of the compound Pien Tze Huang lozenges.

[0054] The present invention adopts surface plasmon resonance method and UPLC method to respectively control the quality of compound Pien Tze Huang lozenges, combines the evaluation of overall efficacy with the precise determination of component composition, and more comprehensively controls the quality of compound Pien Tze Huang lozenges, thus having practical application value.

[0055] 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.

[0056] 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

[0057] Figure 1 Coupling results of TNFR2 protein in CM5

[0058] Figure 2 Results of TNFR2 protein specificity investigation

[0059] Figure 3 Affinity Verification of Physcion 8-O-glucoside on TNFR2 Protein Chip

[0060] Figure 4 Linear relationship diagram of physcion-8-O-glucoside at different cycle numbers

[0061] Figure 5 Relationship between different concentrations of physcion-8-O-glucoside and peak area

[0062] Figure 6 TNFR2 recognizes and recovers binding components of running buffer (A) and compound Pien Tze Huang tablets (B)

[0063] Figure 7 Total ion currents of TNFR2 protein recovery samples in running buffer (A) and Compound Pien Tze Huang Lozenges (B); 1. Isofraxidin; 2. Isochlorogenic acid B; 3. Isochlorogenic acid A; 4. Isochlorogenic acid C; 5. Isoliquiritin; 6. Isoliquiritin; 7. Notoginsenoside R1; 8. Ginsenoside Rh1; 9. Ginsenoside Rb1; 10. Ginsenoside Rd;

[0064] Figure 8 Total ion currents of TLR4 protein recovery samples from running buffer (A) and compound Pien Tze Huang tablets (B); 1. Isofraxidin; 2. Neoliquiritin; 3. Nepetaside; 4. Verbascoside; 5. Isochlorogenic acid B; 6. Isochlorogenic acid A; 7. Isochlorogenic acid C; 8. Isoliquiritin; 9. Liquoricerin; 10. Formononetin;

[0065] Figure 9 Total ion currents of TLR5 protein recovery samples in running buffer (A) and Compound Pien Tze Huang Lozenges (B); 1. Neochlorogenic acid; 2. Chlorogenic acid; 3. Cryptochlorogenic acid;

[0066] Figure 10 Precision test of isochlorogenic acid B on TNFR2 chip (A), precision test of compound Pien Tze Huang lozenges on TNFR2 chip (B), repeatability test (C), and stability test (D);

[0067] Figure 11The test results of Compound Pien Tze Huang Lozenges 2204058 on TNFR2;

[0068] Figure 12 The test results of different batches of Compound Pien Tze Huang lozenges on TNFR2;

[0069] Figure 13 Chromatographic overlay of 18 batches of compound Pien Tze Huang lozenge samples and reference fingerprints R;

[0070] Figure 14 UPLC chromatograms of Compound Pien Tze Huang Lozenges S1 sample (A) and mixed reference substance (B); 1. Neochlorogenic acid; 2. Chlorogenic acid; 3. Cryptochlorogenic acid; 4. Caffeic acid; 8. Ferulic acid; 9. Isofraxidin; 10. Liquiritin; 11. Verbascoside; 12. Isochroogenic acid B; 13. Isochrogenic acid A; 14. Isochrogenic acid C; 15. Angoside C; 17. Isochroside;

[0071] Figure 15 UHPLC spectra of Compound Pien Tze Huang Lozenges S1 sample (A) and mixed reference substance (B). DETAILED DESCRIPTION

[0072] 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 TNFR2 protein (C830) was purchased from Suzhou Jinan Protein Technology Co., Ltd.; recombinant human TLR5 protein (CSB-YP023604HU) was purchased from Wuhan Huamei Bioengineering Co., Ltd.; recombinant human TLR4 protein (10146-H08B) was purchased from Beijing Yiqiao Shenzhou 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 membrane (28-9758-16) were purchased from Cytiva, USA; and compound Pien Tze Huang lozenges were purchased from Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd.

[0073] Example 1 Establishment of a method for quality control of compound Pien Tze Huang buccal tablets based on protein chip fishing for anti-inflammatory active ingredients

[0074] 1. Methods and Results

[0075] In the early stage, network pharmacology analysis showed that Compound Pien Tze Huang Lozenges mainly exerted anti-inflammatory effects by regulating TLP channels and TNF signaling pathways. Therefore, focusing on TNF and TLR-related protein targets, an SPR target chip was constructed to screen the active ingredients of Compound Pien Tze Huang Lozenges and explain the "interaction relationship" between proteins and small molecule ligands.

[0076] 1. Solution Preparation

[0077] 1.1 Protein solution preparation

[0078] TNFR2, TLR4 and TLR5 protein powders (100 μg) stored in a -80°C refrigerator were centrifuged at 14000 rpm for 2 min and prepared with sterile water to a concentration of 50 μg mL -1 The protein solution was divided into 20 μL per tube, one tube was taken for each use, and the rest was sealed and stored in a -80℃ refrigerator.

[0079] 1.2 Preparation of fishing test solution

[0080] Take 0.1 g of compound Pien Tze Huang lozenges, add 10 mL of 60% methanol and ultrasonically treat for 30 min, shake well, let it stand, take the supernatant and centrifuge at 12000 rpm for 10 min, take the supernatant and filter it with a 0.22 μm microporous filter membrane, take 1.0 mL of the filtrate and place it in a 1.5 mL centrifuge tube, centrifuge and concentrate to dryness, then add 100 μL of DMSO to dissolve it, and then add 1900 μL of PBS1× solution and mix well to obtain the compound Pien Tze Huang lozenge test solution in PBS buffer containing 5% DMSO.

[0081] 1.3 Preparation of reference solution

[0082] Take the reference powders of isoflurane, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiosyl isoliquiritin, isoliquiritin, notoginsenoside R1, ginsenoside Rh1, ginsenoside Rb1 and ginsenoside Rd, and add pure methanol to prepare about 1.0 mg mL each. -1 The single reference substance stock solution was further diluted to a concentration of approximately 1.0 μg mL -1 The mixed reference solution I was used for the qualitative identification of TNFR2 fishing components; appropriate amounts of isoflurane, neoliquiritin, nepetaside, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiol isoliquiritin, liquiritigenin, and formononetin reference substance powders were taken and added with pure methanol to prepare about 1.0 mg mL each. -1 The single reference substance stock solution was further diluted to a concentration of approximately 1.0 μg mL -1 The mixed reference solution II is used for qualitative identification of TLR4 fishing components; take appropriate amounts of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid reference powders and add pure methanol to prepare approximately 1.0 mg mL each. -1 The single reference substance stock solution was further diluted to a concentration of approximately 1.0 μg mL -1 The mixed reference solution III is used for the qualitative identification of TLR5 fishing components.

[0083] 2. Construction and verification of target fishing chip

[0084] 2.1 Construction of TNFR2 target fishing chip

[0085] Based on the results of the previous pre-enrichment experiment (protein concentration 50 μg·mL-1, protein pH = 4.0), the TNFR2 protein ligand was coupled to the CM5 chip using the amino coupling method. The specific steps are as follows: first, the chip was activated with an amino coupling reagent (EDC / NHS) solution, and then protein coupling was performed. The protein flow rate was set to 5 μL·min -1 , time for 12 minutes; finally, ethanolamine was used to block the unreacted carboxyl sites on the chip surface for 7 minutes. Figure 1 , the coupling amounts of TNFR2 protein to the three channels of CM5 chip were 14336, 13865, and 14353 RU, respectively. The coupling amounts were sufficient for fishing experiments.

[0086] 2.2 Construction of TLR4 target fishing chip

[0087] Based on previous studies, the construction of the TLR4 protein fishing chip was consistent with that of TNFR2. TLR4 was pre-enriched and coupled at an optimal pH of 4.0. The coupling amount of TLR4 was greater than 15,000 RU, which was in line with expectations and could be used for fishing experiments.

[0088] 2.3 Construction of TLR5 target fishing chip

[0089] The construction of the TLR5 protein fishing chip was similar to that of TNFR2. Protein pre-enrichment and coupling were performed, and the signal values flowing through the chip at different pH values were observed. The signal values at pH = 4.0 and pH = 5.0 were similar, with pH = 4.5 being the optimal. Therefore, a protein buffer solution at pH = 4.5 was used to construct the fishing chip, and the TLR5 protein ligand was coupled to the CM5 chip using the amino coupling method. According to the formula TLR5 target coupling amount RL = Rmax × MWligand / (MWanalyte × Sm), the actual coupling amount of TLR5 protein to the CM5 chip channel was 8512 RU, which was in line with expectations and could be used for fishing experiments.

[0090] 2.4 Target fishing chip methodology validation

[0091] The TNFR2 protein chip is specific; active components bind to it and generate signals, while inactive components do not. Therefore, the constructed TNFR2 protein chip was used for the validation of the target fishing chip methodology. Literature reports indicate that physcion 8-O-glucoside inhibits TNF-α-mediated cytotoxicity and apoptosis in L929 cells, while matrine does not bind to the TNF protein. Therefore, physcion 8-O-glucoside was used as a positive control, and matrine was used as a negative control, in the validation of the target fishing chip methodology.

[0092] 2.4.1 Investigation of TNFR2 chip specificity 5 μM positive control physcion-8-O-glucoside, 5 μM negative control matrine and running buffer were injected into the Biacore T200 system respectively to detect the binding response value of TNFR2 protein and the affinity of physcion-8-O-glucoside to TNFR2 protein. The specific steps are as follows: the positive control was prepared into different concentration gradients (0-5 μM) and injected into Biacore T200, and the flow rate, association time and dissociation time were set to 30 μL·min respectively. -1 , 80s and 180s. The data were then processed and analyzed using Biacore T200 evaluation software, using a 1:1 steady-state affinity model. Figure 2 Matrine and running buffer have similar response values (about 0RU), and the response value of physcion-8-O-glucoside is 27.6RU, which shows that the small molecule ligand has specific binding to the TNFR2 chip. Affinity analysis was performed to further verify the specificity of the TNFR2 chip. The results showed that the K value of physcion-8-O-glucoside binding to TNFR2 protein was 27.6RU. D The value is 2.39 μM, see Figure 3 This further demonstrates that the positive small molecule ligand has specific binding to the TNFR2 chip.

[0093] 2.4.2 Linearity investigation and detection limit of recovery cycles The constructed chip was applied to the active ingredient fishing of Compound Pien Tze Huang Lozenges to further determine the appropriate number of cycles for fishing. The specific steps are as follows: prepare a 5μM physophanol methyl ether-8-O-glucoside reference solution, and recover different numbers of cycles on the TNFR2 chip - 1, 2, 5, 10, 20, 50 and 100 recovery cycles. After nitrogen evaporation, re-dissolve with 100μL of 50% methanol and inject into UPLC-QqQ-MS / MS for detection. The chromatographic peak area and S / N value were calculated by Masslynx software. In the set cycle, the S / N of 1Cycles and 2Cycles were less than 10, and the noise was high. Therefore, at least 5Cycles are required to accurately detect the sample. However, the chemical composition of traditional Chinese medicine is complex, and the chemical properties between the components are still quite different. Therefore, 10Cycles detection was used. The chromatographic peak area (Y) was further linearly regressed with the number of recovery cycles (X). See Figure 4 , the recycling method showed a good linear relationship between 1 and 100 recycling cycles.

[0094] 2.4.3 Saturation investigation and concentration detection limit In the screening of active ingredients of traditional Chinese medicine, too high a concentration may affect the activity of the protein, while too low a concentration is not conducive to detection by LC-QqQ-MS. Appropriate concentration can improve the screening efficiency. Therefore, this setting uses different concentration gradients of positive small molecules to carry out fishing recovery on the TNFR2 chip, including 0.39075, 0.7815, 1.5625, 3.125, 6.25, 12.5, 25, and 50 μM. According to the results of the investigation of the number of recovery cycles, the number of cycles for each concentration is set to 10 times. After the recovered liquid nitrogen is blown dry, it is re-dissolved with 100 μL of 50% methanol and injected into UPLC-QqQ-MS / MS for detection. When the concentration is 0.39075 μM, the S / N is less than 10. It is recommended that the subsequent concentration setting should be greater than 0.39075 μM. The correlation analysis of concentration and peak area is shown in Figure 2. Figure 5 When the concentration was greater than 25 μM, the amount of physcion-8-O-glucoside recovered by SPR tended to be saturated, indicating that the TNFR2 chip was saturated.

[0095] 3. Fishing and identification of active ingredients in compound Pien Tze Huang lozenges

[0096] 3.1 Recovery of fishing components

[0097] The test solution and running buffer of Compound Pien Tze Huang lozenges were injected into the Biacore T200 system respectively. The dissociation reagent was 0.5% TFA solution with a volume of 2 μL. The recovery reagent was 50 mM NH4HCO3 solution with a volume of 10 μL. The flow rate was set to 5 μL min -1 , the injection time was set to 60s, the binding time was set to 180s, the dissociation time was set to 60s, and 10 recovery cycles were set. The results are shown in Figure 6 After the fishing, the fishing sample solutions and Running buffer solutions of different proteins were recovered, blown to dryness with nitrogen, and then re-dissolved in 100 μL of 50% methanol for UPLC-Q-TOF-MS analysis.

[0098] 3.2 Identification of fishing components using UPLC-Q-TOF-MS technology

[0099] Chromatographic conditions: Waters CORTECS UPLC C 18Chromatographic column (2.1mm×100mm, 1.6μm); acetonitrile (B)-0.1% formic acid water (A) was used as the mobile phase for gradient elution (0~0.5min, 5%B; 0.5~5min, 5%B; 5~13min, 5~13%B; 13~17min, 13%B; 17~29min, 13~25%B; 29~31min, 25%B; 31~36min, 25~ 30% B; 36-41 min, 30% B; 41-48 min, 30-35% B; 48-52 min, 35% B; 52-53 min, 35-38% B; 53-56 min, 38-48% B; 56-66 min, 48-57% B; 66-72 min, 57-70% B; 72-78 min, 70-85% B); flow rate: 0.25 mL min -1 , column temperature 45℃; injection volume 2μL.

[0100] Mass spectrometry conditions: Time-of-flight mass spectrometry using an electrospray ion source, negative ion mode, capillary voltage: -3.0 kV, desolvation gas flow: nitrogen, flow rate: 800 L·h -1 , desolvation temperature: 500℃, cone gas flow: nitrogen, cone voltage: 30.0V, flow rate: 50L·h -1 , collision gas: argon, collision energy: 10-55eV, ion source temperature: 120℃. Mass spectrometry data were measured using MS e Acquisition was performed in full scan mode with a data acquisition range of m / z 50–1500. Real-time calibration was performed for leucine enkephalin, with m / z 554.2615 in negative ion mode.

[0101] The different protein fishing samples obtained in "1.2 and 1.3" and the prepared mixtures I, II and III were injected into UPLC-Q-TOF-MS respectively, and the fishing components were identified under the above chromatographic and mass spectrometric conditions. The total ion current is shown in Figures 7-9 A total of 10 components were identified from TNFR2 protein, namely isofraquinone, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiosyl isoliquiritin, isoliquiritin, notoginsenoside R1, ginsenoside Rh1, ginsenoside Rb1 and ginsenoside Rd, and the mass spectrum information table of identification is shown in Table 1; a total of 10 components were identified from TLR4 protein, namely isofraquinone, neoliquiritin, nepetaside, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiosyl isoliquiritin, liquiritigenin and formononetin, and the mass spectrum information table of identification is shown in Table 2; a total of 3 components were identified from TLR5 protein, namely neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, and the mass spectrum information table of identification is shown in Table 3.

[0102] Table 1 TNFR2 protein fishing ingredients in compound Pien Tze Huang lozenges

[0103]

[0104]

[0105] Table 2 TLR4 protein fishing ingredients in compound Pien Tze Huang lozenges

[0106]

[0107] Table 3 TLR5 protein fishing ingredients in compound Pien Tze Huang lozenges

[0108]

[0109] 3.3 Affinity Verification and Molecular Docking

[0110] 3.3.1 Verification of the affinity of the fishing components of Compound Pien Tze Huang Lozenges

[0111] To verify the affinity of each fishing component for the TNFR2, TLR4, and TLR5 microarrays, the components were injected into the SPR system at varying concentration gradients, and the affinity test results for each component were calculated by computer fitting. The affinity results for each fishing component for the TNFR2 microarray showed that the KD values for each fishing component were: isofrazinidine 7.20 μM, isochlorogenic acid B 1.97 μM, isochlorogenic acid A 8.44 μM, isochlorogenic acid C 2.43 μM, apiosyl isoliquiritin 6.03 μM, isoliquiritin 8.78 μM, notoginsenoside R1 1.16 μM, ginsenoside Rh1 1.07 μM, ginsenoside Rb1 1.06 μM, and ginsenoside Rd 8.85 μM. The fishing component with the minimum KD value was ginsenoside Rb1, and the KD for binding to the positive control physcion-8-O-glucoside was 2.39 μM. The KD value of ginsenoside Rb1 was less than that of physcion-8-O-glucoside. The affinity results of each fishing component with TLR4 chip showed that the KD of each fishing component were: isoflurane 7.07 μM, neoliquiritin 4.53 μM, nepetaside 14.97 μM, verbascoside 18.70 μM, isochlorogenic acid B 4.55 μM, isochlorogenic acid A 9.88 μM, isochlorogenic acid C 6.38 μM, isoliquiritin 2.57 μM, glycyrrhizin 0.78 μM and tricholoma 8.82 μM. The component with the lowest KD value was liquiritigenin. The affinity analysis of each component with the TLR5 chip showed that the KD values for each component were: neochlorogenic acid (57.24 μM), chlorogenic acid (9.69 μM), and cryptochlorogenic acid (38.53 μM). Chlorogenic acid was the component with the lowest KD value. See Table 4 for details.

[0112] Table 4 Protein fishing components K D values, molecular docking binding energy and binding sites

[0113]

[0114]

[0115] 3.3.2 Molecular docking

[0116] The 2D structure of the small molecule ligand was obtained through the PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ), and the 2D structure was input into the Chem Office software to generate its 3D structure and saved as a mol2 file. Then, the RCSB PDB database (http: / / www.rcsb.org / ) was used to screen the protein target and the high-resolution crystal structures TNFR2 (PDB:8HLB) and TLR5 (PDB:3J0A) were used as molecular docking receptors. The protein was dehydrated and dephosphorylated using PyMOL software and saved as a PDB file. The compound was energy minimized using Molecular Operating Environment 2019 software, the target protein was pretreated, and the active pocket was found. Finally, MOE 2019 was run for molecular docking. The molecular docking binding results showed that each compound could enter the binding domain of different target proteins, and the molecular docking energy range of the compound and TNFR2 target was -7.85 to -4.49 kcal·mol -1 Ginsenoside Rb1 has the strongest binding energy with TNFR2, which is -7.85 kcal·mol -1 It can form hydrogen bond interactions with multiple binding sites of TNFR2 receptor (Tyr61, Gln109, Thr97, Cys104, Lys108, Glu110), residue Ile95 forms hydrophobic interactions with ginsenoside Rb1, and residue Glu110 forms electrostatic interactions with ginsenoside Rb1; K D The value was 1.97 μM (its K D The value is also smaller than that of physcion-8-O-glucoside), and the docking binding energy with TNFR2 is -6.37 kcal·mol -1 , Gly111, Lys108, Arg113, Gln82, Ser73, Cys74, Arg94 and Cys78 residues in TNFR2 form hydrogen bond interactions with isochlorogenic acid B, and residue Arg77 forms an electrostatic interaction with isochlorogenic acid B. In the TLR4 protein fishing experiment, the K D The value ranges from 0.78 to 18.70 μM, among which K DThe smallest value is liquiritigenin (0.78 μM). In the docking with TLR4 receptor, the molecular docking energy range of the compound and TLR4 target is -5.50 to -4.58 kcal·mol -1 The binding of liquiritin to TLR4 target protein is the strongest, with a docking energy of -5.49 kcal·mol -1 , Asp395, Lys388, Phe387, Gln64 and Ser386 residues in TLR4 target protein form hydrogen bond interactions with liquiritigenin. In the TLR5 protein fishing experiment, the K D The values ranged from 9.69 to 57.24 μM, and K D The smallest value was for chlorogenic acid (9.69 μM). In the docking with TLR5 target protein, the molecular docking energy range was -6.15 to -5.86 kcal·mol -1 Chlorogenic acid has the strongest binding to TLR5 target protein, with a binding energy of -6.15 kcal·mol -1 Ser625, Ser641, and Glu631 residues in the TLR5 target protein form hydrogen bond interactions with chlorogenic acid, and residue Phe616 forms a hydrophobic interaction with chlorogenic acid, suggesting that the affinity test K of Compound Pien Tze Huang Lozenges can be verified by molecular docking binding energy strength. D Value result.

[0117] 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 TNFR2 protein, TLR4 protein and TLR5 protein targets from the complex system of Compound Pien Tze Huang Lozenges. These active ingredients revealed that Compound Pien Tze Huang Lozenges can exert anti-inflammatory effects through TNFR2, TLR4 and TLR5 protein targets, laying the foundation for the biological effect evaluation of Compound Pien Tze Huang Lozenges.

[0118] The above-mentioned verified method of targeting the active ingredients of Compound Pien Tze Huang Lozenges based on different protein targets is as follows:

[0119] 1) Construction of protein chip:

[0120] TNFR2 protein, TLR4 protein and TLR5 protein were respectively dissolved in sterile water to obtain 500 μg / ml TNFR2 protein mother solution, TLR4 protein mother solution and TLR5 protein mother solution;

[0121] Take 10 μl of TNFR2 protein stock solution and TLR4 protein stock solution respectively, add 90 μl of 10 mM pH 4.0 sodium acetate buffer and mix well to obtain 50 μg / ml TNFR2 protein solution and TLR4 protein solution;

[0122] Take 10 μl of TLR5 protein stock solution, add 90 μl of 10 mM pH 4.5 sodium acetate buffer and mix well to obtain 50 μg / ml TLR5 protein solution;

[0123] Start the surface plasmon resonance instrument, set channel 1 as the blank unit and channel 2 as the coupling unit,

[0124] 100 μl of TNFR2, TLR4, and TLR5 protein solutions were respectively taken and amino-coupled to a CM5 chip. The coupling parameters were: contact 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.

[0125] 2) Preparation of fishing test solution

[0126] Take 0.1 g of Compound Pien Tze Huang lozenges, add 10 mL of 60% methanol, and sonicate for 30 min. Shake well, let it stand, and take the supernatant, centrifuge at 12000 rpm for 10 min. Filter the supernatant with a 0.22 μm microporous filter membrane, take 1.0 mL of the filtrate, place it in a 1.5 mL centrifuge tube, concentrate it to dryness by centrifugation, add 100 μL of DMSO to dissolve it, and then add 1900 μL of PBS 1× solution and mix well.

[0127] 3) Fishing

[0128] The test solution was injected into the surface plasmon resonance instrument loaded with TNFR2 protein, TLR4 protein and TLR5 protein chips for fishing. The fishing conditions were as follows: mobile phase: PBS buffer solution containing 5% DMSO; sample flow rate: 5 μL·min -1 Injection time: 60 s; Binding time: 180 s; Dissociation time: 60 s; Dissociation reagent: 0.5% TFA solution, Dissociation reagent volume: 2 μL, Dissociation reagent incubation time: 20 s; Recovery reagent: 50 mM NH4HCO3 solution, Recovery reagent volume: 10 μL; Number of cycles: 10 times;

[0129] 4) UPLC-Q-TOF-MS qualitative detection of the collected liquid after fishing

[0130] a: Preparation of reference solution: Take isoflurane, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiose isoliquiritin, isoliquiritin, notoginsenoside R1, ginsenoside Rh1, ginsenoside Rb1 and ginsenoside Rd reference substances, add methanol to dissolve as the reference solution for qualitatively targeting the active ingredient of TNFR2 protein;

[0131] Take isoflurane, neoliquiritin, nepetaside, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiosyl isoliquiritin, liquiritigenin and formononetin reference substances, add methanol to dissolve as reference solution for qualitative targeting of TLR4 protein active ingredients;

[0132] Take neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid reference substances and dissolve them in methanol as reference solution for qualitatively targeting the active ingredients of TLR5 protein;

[0133] b: Take the recovered fishing component solution after fishing, dry it, add 50% methanol to dissolve it, and inject the dissolved solution and the reference solution obtained in A3 into UPLC-Q-TOF-MS for detection:

[0134] Chromatographic conditions: Waters CORTECS UPLC C 18 Chromatographic column (2.1mm×100mm, 1.6μm); acetonitrile (B)-0.1% formic acid water (A) was used as the mobile phase for gradient elution (0~0.5min, 5%B; 0.5~5min, 5%B; 5~13min, 5~13%B; 13~17min, 13%B; 17~29min, 13~25%B; 29~31min, 25%B; 31~36min, 25~ 30% B; 36-41 min, 30% B; 41-48 min, 30-35% B; 48-52 min, 35% B; 52-53 min, 35-38% B; 53-56 min, 38-48% B; 56-66 min, 48-57% B; 66-72 min, 57-70% B; 72-78 min, 70-85% B); flow rate: 0.25 mL min -1 , column temperature 45°C; injection volume 2 μL;

[0135] Mass spectrometry conditions: Time-of-flight mass spectrometry using an electrospray ion source, negative ion mode, capillary voltage: -3.0 kV, desolvation gas flow: nitrogen, flow rate: 800 L·h -1 , desolvation temperature: 500℃, cone gas flow: nitrogen, cone voltage: 30.0V, flow rate: 50L·h -1 , collision gas: argon, collision energy: 10-55eV, ion source temperature: 120℃. Mass spectrometry data were measured using MS e Acquisition was performed in full scan mode with a data acquisition range of m / z 50–1500. Real-time calibration was performed for leucine enkephalin, with m / z 554.2615 in negative ion mode.

[0136] Example 1 SPR technology was used to screen out the anti-inflammatory active ingredients of Compound Pien Tze Huang Lozenges at different protein targets. The following biological effect determination method was established to control the quality of Compound Pien Tze Huang Lozenges in a simpler and more convenient way.

[0137] Example 2 Establishment of a method for quality control of compound Pien Tze Huang buccal tablets based on biological effects

[0138] 1. Establishment of Q-biomarker methodology for Compound Pien Tze Huang lozenges

[0139] Since the TNFR2 protein ranked high in the network pharmacology screening and the components of the Compound Pien Tze Huang lozenges bind well to the protein, the TNFR2 protein was used in the methodological investigation of the biological effect detection of the Compound Pien Tze Huang lozenges. Since the affinity verification results in Example 1 showed that the content of isochlorogenic acid B in the Compound Pien Tze Huang lozenges was high (derived from the main drug Wedelia chrysantha) and the KD value was small, the methodological investigation of the biological effect detection of the Compound Pien Tze Huang lozenges used isochlorogenic acid B as a positive control.

[0140] 1.1 Precision test The same positive control substance isochlorogenic acid B solution was injected into Biacore T200 (with a built-in TNFR2 protein chip) for analysis 6 times. The RU values obtained by the 6 injections were 52.9, 52.4, 52.4, 52.3, 52.3, and 52.0, respectively, with an RSD of 0.6%. The same batch of compound Pien Tze Huang lozenges (batch number 2204058) was taken and the test solution was prepared according to the method under "1.2 Preparation of test solution for fishing" in Example 1. The sample was injected and the RU values obtained by the 6 injections were 127.7, 127.0, 126.5, 125.1, 124.0, and 123.2, respectively, with an RSD of 1.4%. Figure 10 The results showed that the precision of the instrument was good.

[0141] 1.2 Repeatability test Six samples of the same batch of compound Pien Tze Huang lozenges (batch number 2204058) were weighed in parallel, and sample solutions were prepared according to the method under "1.2 Preparation of test solution for fishing" in Example 1. The RU values of the six samples of compound Pien Tze Huang lozenges were 145.7, 144.3, 138.8, 138.0, 132.2, and 131.2, respectively, with an RSD of 4.3%. The results showed that the method had good repeatability.

[0142] 1.3 Stability test The same batch of compound Pien Tze Huang lozenges (batch number 2204058) were taken, and sample solutions were prepared according to the method under "1.2 Preparation of test solution for fishing" in Example 1. The samples were injected into Biacore T200 (with a built-in TNFR2 protein chip) for analysis at 0, 2, 4, 8, 12 and 24 h, respectively. The RU values obtained by 6 injections were 126.1, 128.2, 130.8, 123.2, 136.2, and 122.1, respectively, and the RSD was 4.1%, indicating that the test solution was stable within 24 h.

[0143] 2 SPR determination of different batches of Compound Pien Tze Huang lozenges

[0144] Different batches of compound Pien Tze Huang buccal tablets were taken, and sample solutions were prepared according to the method under "1.2 Preparation of fishing test solution" in Example 1. The obtained samples were injected into Biacore T200 (with a built-in TNFR2 protein chip) for analysis together with the positive component isochlorogenic acid B of the compound Pien Tze Huang buccal tablets, the negative component leonurin and the running buffer. The ratio of the RU value of the compound Pien Tze Huang buccal tablets to the RU value of the positive component of the different batches is shown in Table 1. The results of the above solution binding experiment with TNFR2 protein are shown in Table 1. Figures 11-12 The ratios of the RU values of Compound Pien Tze Huang lozenges 2204058, 2104035, 2201003, 20210401-1, 20210402-1, T2304017-3, T2304019-5, T2304021-3, T2304005-C, T2304006-C, and T2304007-C to the RU value of the positive component isochlorogenic acid B were 4.9, 4.2, 4.7, 13.2, 12.4, 5.8, 10.1, 5.8, 5.0, 12.9, and 8.9, respectively, all greater than 1.

[0145] Table 1 RU values and ratios of compound Pien Tze Huang lozenge samples and isochlorogenic acid B

[0146]

[0147] The Q-biomarker methodology of the above-mentioned Compound Pien Tze Huang Lozenges was validated and the RU value of the Compound Pien Tze Huang Lozenges on the TNFR2 target chip was detected. The precision RSD of the positive control isochlorogenic acid B was 0.6%, the sample precision RSD was 1.4%, the repeatability RSD was 4.3%, and the stability RSD was 4.1%. The precision, repeatability and stability were good, indicating that the constructed Q-biomarker methodology can be used for the detection of Compound Pien Tze Huang lozenges samples. The RU values of each batch of samples were in the range of 118.4 to 403.3, and the RU values of isochlorogenic acid B were in the range of 27.8 to 30.5, respectively. The ratios were between 4.2 and 13.2, all greater than 1. Based on this, the detection method for the biological effect of Compound Pien Tze Huang Lozenges was determined to be:

[0148] 1) Protein chip construction: TNFR2 protein was dissolved in sterile water to obtain a 500 μg / ml protein stock solution. 10 μl of the protein stock solution was added to 90 μl of 10 mM pH 4.0 sodium acetate buffer and mixed to obtain a 50 μg / ml 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 TNFR2 protein solution was amino-coupled to a CM5 chip using the following parameters: contact 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 Test Solution: 0.1 g of Compound Pien Tze Huang lozenge sample was placed in a 25 mL Erlenmeyer flask, 10 mL of 60% methanol was added, the sample was sealed and weighed, ultrasonically extracted for 30 min (50 kHz, 350 W), and then weighed again. The sample was supplemented with 60% methanol to make up for the loss in weight. The sample was centrifuged at 14,000 rpm for 10 min, 2 mL of the supernatant was accurately aspirated, concentrated by centrifugation, evaporated to dryness, and redissolved in 200 μL of DMSO. The sample was filtered through a 0.22 μm microporous membrane, 10 μL of the sample was accurately aspirated, and 190 μL of PBS 1× solution was added and mixed.

[0152] 3) Preparation of Reference Solution: Accurately weigh isochlorogenic 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.

[0153] 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);

[0154] SPR detection conditions: association time: 60 s; flow rate: 30 μl / min; dissociation time: 60 s; temperature: 25° C.; running buffer: 5% DMSO in PBS.

[0155] 5) Calculate the ratio of the RU values of the Compound Pien Tze Huang Lozenges to that of isochlorogenic acid B. When the ratio is greater than or equal to 1, the biological effect test of the Compound Pien Tze Huang Lozenges is qualified.

[0156] Example 1 SPR technology was used to screen out the anti-inflammatory active ingredients of Compound Pien Tze Huang Lozenges at different protein targets. The following UPLC method was established to perform fingerprint analysis on the anti-inflammatory active ingredients to further comprehensively control the quality of Compound Pien Tze Huang Lozenges.

[0157] Example 3: Establishment of a method for quality control of compound Pien Tze Huang lozenges by combining fingerprint analysis with multi-component determination

[0158] 1. Drug testing

[0159] 18 batches of compound Pien Tze Huang lozenge samples were purchased from Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd.

[0160] 2 Methods and Results

[0161] 2.1 Chromatographic conditions

[0162] Using Welch UltimateAQ-C 18 Chromatographic column (2.1 mm × 150 mm, 1.8 μm); acetonitrile (B)-0.1% formic acid water (A) as the mobile phase for gradient elution (0-13 min, 5-13% B; 13-16 min, 13-15.5% B; 16-25 min, 15.5-21% B; 25-28 min, 21% B; 28-32 min, 21-22% B; 32-41 min, 22-30% B); flow rate 0.25 mL min -1 ; Column temperature 30℃; Injection volume 2μL; Detection wavelength 330nm.

[0163] 2.2 Preparation of reference solution

[0164] Appropriate amounts of reference substance powders of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid, isoflurane, liquiritin, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoside C, and isoliquiritin were accurately weighed and added with pure methanol to prepare reference substances with concentrations of 0.630, 0.824, 0.720, 1.141, 1.320, 1.185, 1.760, 0.545, 0.946, 1.604, 0.879, 3.924, and 1.360 mg mL, respectively. -1 The single reference substance mother solution was accurately weighed and placed in a 10 mL volumetric flask, and 50% methanol was added to make up the volume to obtain a mixed reference substance solution I of chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid, isofraquinone, liquiritin, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoside C, and isoliquiritin, which was used to identify the common peaks in the fingerprint of Compound Pien Tze Huang lozenges. The mass concentrations of the mixed reference substance solutions I were 6.300, 8.240, 7.200, 2.282, 2.640, 4.740, 35.200, 2.180, 18.920, 8.020, 17.580, 15.696, and 6.800 μg mL -1 Then accurately measure the above-mentioned reference substance stock solutions (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid, isoflurane, liquiritin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoside C, isoliquiritin) and place them in a 100 mL volumetric flask. Add 50% methanol to make up the volume, so that the mass concentrations of the above components are 15.750, 20.600, 18.000, 4.564, 6.600, 5.925, 44.000, 18.920, 16.040, 21.975, 15.696, and 6.800 μg mL, respectively. -1 The mixed reference solution II was used as the linear mother solution for content determination of Compound Pien Tze Huang lozenges.

[0165] 2.3 Preparation of test solution

[0166] Grind the Compound Pien Tze Huang buccal tablets (without coating) into fine powder and mix thoroughly. Accurately weigh 2.0 g of powder and place it in a stoppered bottle. Accurately transfer 25 mL of 60% methanol solution into the powder and weigh it. Ultrasonicate for 30 min (power 250 W, frequency 50 kHz). Let cool and weigh it again. Make up the lost weight with the extracted solvent. Shake well and centrifuge at 12000 rpm for 10 min. Filter through a 0.22 μm filter membrane and take the filtrate to obtain the Compound Pien Tze Huang buccal tablet test solution.

[0167] 2.4 Study on the fingerprint of compound Pien Tze Huang lozenges

[0168] 2.4.1 Precision test

[0169] Accurately pipette 2 μL of the compound Pien Tze Huang lozenge solution from batch S1 and perform six consecutive injections according to the chromatographic conditions in "2.1." Peak 12 was selected as the reference peak because its single peak area accounts for a large proportion of the total peak area, its retention time (26.43 min) is located in the middle of the spectrum, and it exhibits high responsiveness and good resolution. The relative retention times and relative peak areas of the common peaks were calculated. Based on the established method, the relative retention time RSD was ≤0.16%, and the relative peak area RSD was ≤1.54%, indicating good method precision.

[0170] 2.4.2 Repeatability test

[0171] Grind the S1 batch sample into fine powder, accurately weigh 6 portions to prepare the test solution, and inject and analyze under the established chromatographic conditions. Take peak 12 as the reference peak, calculate the relative retention time and relative peak area RSD of the common peak, and the relative retention time RSD is ≤0.23%, and the relative peak area RSD is ≤2.93%, indicating that the repeatability of each component is good.

[0172] 2.4.3 Stability test

[0173] Accurately aspirate 2 μL of the test solution of Compound Pien Tze Huang lozenges from batch S1, and inject the sample for analysis at 0, 2, 4, 8, 12, and 24 h, respectively. Taking peak 12 as the reference peak, calculate the relative retention time and RSD of the relative peak area of the common peak. According to the established method, the relative retention time RSD ≤ 0.51%, and the relative peak area RSD ≤ 3.34%, indicating that the test solution is stable within 24 h.

[0174] 2.4.4 Establishment of UPLC fingerprint of Compound Pien Tze Huang lozenges

[0175] Accurately aspirate 2 μL of each test solution of Compound Pien Tze Huang Lozenges from S1 to S18, perform sample injection and analysis according to the chromatographic conditions under "2.1", and record the chromatogram of each sample. The obtained chromatographic data were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A Edition)", with S1 as the reference spectrum, using the median method, the time window set to 0.1 min, the spectrum spacing set to 30, and the chromatographic peaks were automatically matched. The chromatographic peaks with stable peaks and good separation were marked as common peaks, and the chromatographic peaks that could not appear stably due to the change of the mobile phase gradient were eliminated. The chromatographic overlays and reference fingerprints R of 18 batches of Compound Pien Tze Huang Lozenges were generated. A total of 18 common peaks were calibrated. The UPLC chromatographic overlays of 18 batches of Compound Pien Tze Huang Lozenges and the reference fingerprints R of Compound Pien Tze Huang Lozenges are shown in Figure 2. Figure 13 After comparison with mixed reference solution I, a total of 13 chromatographic peaks were identified, among which the retention time and spectral behavior of the compound Pien Tze Huang lozenge sample were consistent with those of the 13 reference substances, as shown in the chromatogram. Figure 14 , spectrum diagram see Figure 15, respectively: 1. neochlorogenic acid (7.98min); 2. chlorogenic acid (12.03min); 3. cryptochlorogenic acid (12.93min); 4. caffeic acid (13.44min); 8. ferulic acid (20.70min); 9. isoflurane (21.29min); 10. glycyrrhizin (22.22min); 11. verbascoside (24.31min); 12. isochlorogenic acid B (26.43min); 13. isochlorogenic acid A (27.18min); 14. isochlorogenic acid C (29.66min); 15. angoside C (30.70min); 17. isoliquiritin (33.84min).

[0176] 2.4.5 Similarity Analysis

[0177] The “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A Version)” was used to import the AIA files of 18 batches of compound Pien Tze Huang lozenge samples for similarity evaluation and calculate the similarity, see Table 5. The similarity results of each batch of Compound Pien Tze Huang lozenges were 0.832-0.999, and the similarities between each batch of Compound Pien Tze Huang lozenges and the control fingerprint R were 0.992, 0.996, 0.988, 0.992, 0.992, 0.994, 0.991, 0.994, 0.907, 0.908, 0.936, 0.987, 0.986, 0.985, 0.989, 0.991, 0.992, and 0.992, all of which were greater than 0.900, indicating that the similarity between batches of Compound Pien Tze Huang lozenges was high, and the established fingerprint method can be used for the overall quality control of Compound Pien Tze Huang lozenges.

[0178]

[0179] 2.5 Determination of the contents of 12 ingredients in Compound Pien Tze Huang lozenges

[0180] 2.5.1 Linearity and Range

[0181] The mixed reference solution II prepared by the method under "2.2" was diluted stepwise with 50% methanol to form a series of reference mixed solutions with gradient concentrations. The peak areas were determined according to the chromatographic conditions under "2.1". The peak areas (Y) of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid, isoflurane, liquiritin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoside C and isoliquiritin were used to perform linear regression on the mass concentration (X) of each analyte. The standard curve was drawn to obtain the regression equation and correlation coefficient (r). The signal-to-noise ratio (S / N) was approximately equal to 10 as the limit of quantification (LOQ), and the S / N was approximately equal to 3 as the limit of detection (LOD). The results are shown in Table 6.

[0182] Table 6 Regression equations, linear ranges, correlation coefficients, detection limits, and quantification limits of the 12 components

[0183]

[0184] 2.5.2 Precision test

[0185] Accurately pipette 2 μL of mixed reference solution II and inject the sample six times within 1 day (n=6) and repeat the injection three times per day for 3 consecutive days (n=9) under the chromatographic conditions of "2.1". The peak areas of the 12 components were measured, and the RSDs of the intra-day and inter-day precision were calculated. The results are shown in Table 7, indicating that the instrument has good precision.

[0186] 2.5.3 Stability test

[0187] Take the S1 batch of compound Pien Tze Huang lozenges and prepare the test solution according to the method under "2.3". The samples were injected and measured according to the chromatographic conditions of "2.1" at 0, 2, 4, 8, 12, and 24 hours respectively. The peak areas of 12 components were measured and the RSD values of the peak areas were calculated. The results are shown in Table 3, indicating that the test solution was stable within 24 hours.

[0188] 2.5.4 Repeatability test

[0189] Six samples of the same batch of Compound Pien Tze Huang lozenges (batch S1) were accurately weighed and the test solutions were prepared according to the method under "2.3". The samples were injected and determined according to the chromatographic conditions under "2.1". The contents of 12 compounds and their RSDs were calculated based on the standard curve. The results are shown in Table 7, indicating that the method had good reproducibility.

[0190] Table 7 Precision, stability and repeatability test results of 12 components

[0191]

[0192] 2.5.5 Recovery rate test

[0193] Accurately weigh 6 samples of Compound Pien Tze Huang lozenges (batch S1) with known content under "2.5.4", about 1.0 g, and add 12 reference substances at an approximate 1:1 ratio according to the content results of the repeatability determination. Prepare the test solution according to the method under "2.3". Inject the sample according to the chromatographic conditions under "2.1". Record the peak areas and substitute them into the standard curve to calculate the contents of the 12 components to be tested. Calculate the recovery rate, which is between 99% and 103%, with an RSD of <4.36%.

[0194] 2.5.6 Sample content determination

[0195] Take samples of Compound Pien Tze Huang lozenges from batches S1 to S18, and analyze them separately according to the chromatographic conditions under "2.1". Calculate the content in the samples, see Table 8.

[0196]

[0197] 3 Discussions

[0198] 3.1 Optimization of chromatographic conditions and extraction process

[0199] 3.1.1 Investigation of mobile phase

[0200] This experiment investigated the elution effects of different mobile phase systems: acetonitrile-0.1% formic acid water, acetonitrile-water, methanol-water, and methanol-0.1% formic acid water. Methanol-water and methanol-0.1% formic acid water had weak elution abilities, fewer chromatographic peaks, and poor peak shapes. Acetonitrile-water and acetonitrile-0.1% formic acid water had comparable chromatographic peak separation performance and stable baselines. However, adding an appropriate amount of formic acid to the mobile phase system could improve the peak shape. Therefore, acetonitrile-0.1% formic acid water was used as the mobile phase in this experiment.

[0201] 3.1.2 Examination of chromatographic columns

[0202] Inspecting the Welch Ultimate XB-C 18 (4.6×150nm, 2.7μm), Welch Ultimate AQ-C 18 (2.1×150nm,1.8μm), Agilent Infinitylab EC-C 18 (2.1×150nm,1.9μm), Welch Xtimate C 18 (2.1×150nm, 1.8μm) and Welch Ultimate XB-C 18 The effects of five chromatographic columns (2.1×150nm, 1.8μm) on the separation of components, including Welch Ultimate XB-C 18 (4.6×150nm, 2.7μm) and Welch Ultimate XB-C 18 (2.1×150nm, 1.8μm) poor peak shape, low column efficiency, Welch Ultimate AQ-C 18 (2.1×150nm,1.8μm), Agilent Infinitylab EC-C 18 (2.1×150nm,1.9μm), Welch Xtimate C 18The separation effect of the chromatographic column (2.1×150nm, 1.8μm) is comparable, with sharp peaks and high column efficiency. After comparison and optimization, the Welch UltimateAQ-C 18 (2.1×150nm,1.8μm) can effectively and stably separate cryptochlorogenic acid from caffeic acid, ferulic acid and adjacent chromatographic peaks, so it was used as the chromatographic column in this experiment.

[0203] 3.1.3 Investigation of injection volume

[0204] Under the established chromatographic conditions, the effects of different injection volumes (1 μL, 2 μL, and 3 μL) on the separation were investigated. No significant changes in the chromatographic peak shapes were observed at injection volumes of 1 to 3 μL, and the system suitability parameters met the analytical requirements, demonstrating the robustness of the method.

[0205] 3.1.4 Investigation of flow rate

[0206] Three different flow rates (0.20 mL min -1 , 0.25mL·min -1 , 0.30mL·min -1 ) The sample was injected and tested under optimized gradient conditions to analyze the effect of different flow rates on the separation of each chromatographic peak. -1 ~0.30mL·min -1 The changes within the range can meet the requirements of separation analysis. With the increase of flow rate, the retention time shifts forward and the separation of some chromatographic peaks decreases, but the changes in chromatographic parameters are not obvious, indicating that the method has good durability.

[0207] 3.1.5 Column temperature inspection

[0208] Under established gradient conditions, the effects of varying column temperatures (25°C, 30°C, and 35°C) on chromatographic peak separation performance were investigated. While the retention times of most compounds shifted with increasing column temperature, the symmetry factor, number of theoretical plates, and resolution of the individual components remained unaffected, demonstrating that each chromatographic peak met the separation and analysis requirements under varying column temperatures.

[0209] 3.1.6 Investigation of Maximum Absorption Wavelength

[0210] The maximum absorption wavelength (λ max ), the results showed that there were fewer chromatographic peaks at 237nm, 254nm, and 273nm, and the baseline was unstable. The chemical components at 300nm, 310nm, 320nm, and 330nm were relatively rich. At 330nm, the chromatographic peaks were well separated, the peak capacity was large, and the baseline was the most stable. Therefore, 330nm was selected as the detection wavelength.

[0211] 3.1.7 Investigation of extraction solvent

[0212] The ultrasonic extraction efficiencies of 20%, 40%, 60%, 80%, and 100% methanol were compared. The results are shown in the attached table. The extraction efficiencies of 20%, 40%, and 100% methanol were lower, while those of 60% and 80% methanol were comparable. From the perspective of solvent conservation (methanol), 60% methanol was the optimal extraction solvent.

[0213] 3.1.8 Investigation of extraction time

[0214] The ultrasonic extraction efficiency was compared at 15 min, 30 min, 45 min and 60 min of ultrasonic extraction (power 250 W, frequency 50 kHz). The results are shown in the attached table. There was no significant change in the extraction efficiency after 30 min of ultrasonic extraction, indicating that the extraction efficiency of 60% methanol ultrasonic extraction for 30 min basically reached the highest level.

[0215] 3.1.9 Investigation of extraction solvent dosage

[0216] The extraction efficiencies of 4, 12.5, and 25 times the extraction volume were compared. The results are shown in the attached table. The results show that the extraction efficiency is low when the extraction volume is 4, and the extraction efficiencies are comparable when the extraction volumes are 12.5 and 25. However, when the extraction volume is 12.5, the responsiveness of each chromatographic peak is better, so the extraction volume of 12.5 was finally selected.

[0217] 3.2 Fingerprint results analysis

[0218] After investigation of fingerprint methodology, the UHPLC fingerprint of Compound Pien Tze Huang lozenges was established. The similarity R between the control fingerprints of 18 batches of Compound Pien Tze Huang lozenges was greater than 0.900, indicating that the similarity between batches of Compound Pien Tze Huang lozenges was high, and the quality of Compound Pien Tze Huang lozenges could be objectively evaluated.

[0219] 3.3 Selection and Limits of Quantitative Indicator Components

[0220] In this study, the contents of 12 chemical components in Compound Pien Tze Huang lozenges were determined by ultrahigh-performance liquid chromatography (UHPLC). Evaluation criteria were selected based on: 1. UPLC-QqQ-MS / MS technology combined with chemical pattern recognition analysis identified differential biomarkers for Compound Pien Tze Huang lozenges; 2. SPR technology was used to screen the active components of Compound Pien Tze Huang lozenges at different protein targets. These active components revealed that Compound Pien Tze Huang lozenges may exert anti-inflammatory effects through protein targets such as TNFR2, TLR4, and TLR5, necessitating effective content determination. Neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isofraquinone, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and isoliquiritin all bind to proteins to exert anti-inflammatory activity. Isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C were the primary differential biomarkers in Compound Pien Tze Huang lozenges (VIP values > 1.5), and therefore these components were included in the UHPLC content determination criteria. Liquiritin is a dihydroflavonoid compound, and isoliquiritin is a chalcone compound. Under certain conditions, the two components will convert into each other. Based on the UPLC-QqQ-MS / MS content determination results, liquiritin causes a difference marker between batches of Compound Pien Tze Huang lozenges. Therefore, liquiritin is also included in the content determination indicators.

[0221] After methodological investigation, a UHPLC method was established to determine the contents of 12 chemical components in Compound Pien Tze Huang lozenges. The highest content of the components in Compound Pien Tze Huang lozenges was liquiritin, with a content of 0.0974-0.3167 mg·g -1 The contents of the potential active ingredients of SPR fishing, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C, were second, and the relationship between their contents was isochlorogenic acid C>isochlorogenic acid B>isochlorogenic acid A, with the contents ranging from 0.0529 to 0.2185 mg·g -1 , 0.0389~0.1641mg·g -1 , 0.0293~0.1397mg·g -1 According to the national drug standard for Compound Pien Tze Huang Lozenges, each tablet (0.5g) of Compound Pien Tze Huang Lozenges contains scutellaria baicalensis and isoflurane (C 11 H 10 O5) should not be less than 6μg, that is, it should be greater than 12μg·g -1 The contents of 18 batches of compound Pien Tze Huang lozenges all met the requirements.

[0222] 4 Summary

[0223] The present invention adopts UHPLC fingerprint and multi-component content determination method to analyze compound Pien Tze Huang lozenges. The method is fast and simple, and can lay a foundation for improving the chemical quality standard of compound Pien Tze Huang lozenges.

Claims

1. A quality control method for compound Pien Tze Huang buccal tablets, characterized in that: It uses surface plasmon resonance to target protein active ingredients in compound Pien Tze Huang lozenges; the protein is TNFR2 protein, TLR4 protein and / or TLR5 protein; When the protein is TNFR2 protein, the active ingredients fished out are isofraquinone, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apigenin isoliquiritin, isoliquiritin, notoginsenoside R1, ginsenoside Rh1, ginsenoside Rb1 and ginsenoside Rd; When the protein is TLR4 protein, the activities fished out are isofraquinone, neoliquiritin, nepetaside, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiol isoliquiritin, liquiritigenin and formononetin; When the protein is TLR5 protein, the activities detected are neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid.

2. The quality control method according to claim 1, characterized in that: The fishing comprises the following steps: A1. Constructing a protein chip: Dissolve TNFR2 protein, TLR4 protein or TLR5 protein in water, then dilute with sodium acetate buffer to a 30-50 μg / ml protein solution. Take the protein solution and couple it with the sensor chip. A2 Preparation of fishing test solution: Take compound Pien Tze Huang lozenges, add 60% methanol solution to extract, centrifuge the extract, filter the supernatant, dry the filtrate, add DMSO to re-dissolve, and then add 1× PBS solution to mix. 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 were as follows: mobile phase: PBS buffer solution containing 5% DMSO; sample flow rate: 5 μL min -1 ; Injection time: 60s; Association time: 180s; Dissociation time: 60s; Dissociation reagent: 0.5% TFA solution, dissociation reagent volume: 2 μL, dissociation reagent incubation time: 20 s; recovery reagent: 50 mM NH4HCO3 solution, recovery reagent volume: 10 μL; number of cycles: 10 times; A4 uses UPLC-Q-TOF-MS method to perform qualitative detection on the fishing component recovery liquid obtained in step A3.

3. The quality control method according to claim 1, wherein: In step A1, the concentration of the sodium acetate buffer is 10 mM, and the pH is 4-4.5; the protein solution is 50 μg / ml; the coupling is amino coupling; the parameters of the amino coupling are: sensor chip model: CM7 or CM5, coupling reagents: EDC and NHS, protein solution flow rate: 5 μl / min, protein contact time: 720 s, temperature: 25°C, blocking reagent: ethanolamine, and blocking time: 7 min; And / or: the mass volume ratio of the compound Pien Tze Huang lozenges in step A2 to the 60% methanol solution is 0.1 g: 5-25 ml; the extraction is ultrasonic extraction, the time is 20-60 min; the centrifugation speed is 5000-25000 r, the time is 5-25 min; the volume ratio of the filtrate, DMSO and PBS 1× solution is 0.5-1.5 mL: 100 μL: 1900 μL.

4. The quality control method according to claim 3, characterized in that: When the protein solution in step A1 is a TNFR2 protein solution or a TLR4 protein solution, the pH value of the sodium acetate buffer is 4; when the protein solution is a TLR5 protein solution, the pH value of the sodium acetate buffer is 4.5; the sensor chip model is CM5, the volume ratio of the protein solution, EDC and NHS, and ethanolamine is 100:200:140; the volume ratio of the EDC and NHS is 1:

1.

5. The quality control method according to claim 1, wherein: The qualitative detection in step A4 includes the following steps: ① Preparation of reference solution: Take isoflurane, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiose isoliquiritin, isoliquiritin, notoginsenoside R1, ginsenoside Rh1, ginsenoside Rb1 and ginsenoside Rd reference substances, add methanol to dissolve as reference solution for qualitative targeting of TNFR2 protein active ingredients; And / or: take isoflurane, neoliquiritin, nepetaside, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, apiosyl isoliquiritin, liquiritigenin and formononetin reference substances, add methanol to dissolve as reference substance solution for qualitatively targeting TLR4 protein active ingredients; And / or: Take neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid reference substances and dissolve them in methanol as reference solution for qualitatively targeting the active ingredient of TLR5 protein; ② The recovered fishing component solution was dried and dissolved in 50% methanol. The dissolved solution and the reference solution obtained in step ① were injected into UPLC-Q-TOF-MS for detection respectively; Chromatographic conditions were as follows: chromatographic column: C18 column; mobile phase: acetonitrile-0.1% formic acid water; gradient elution program: 0-0.5 min, 5% acetonitrile; 0.5-5 min, 5% acetonitrile; 5-13 min, 5-13% acetonitrile; 13-17 min, 13% acetonitrile; 17-29 min, 13-25% acetonitrile; 29-31 min, 25% acetonitrile; 31-36 min, 25-3 0% acetonitrile; 36-41 min, 30% acetonitrile; 41-48 min, 30-35% acetonitrile; 48-52 min, 35% acetonitrile; 52-53 min, 35-38% acetonitrile; 53-56 min, 38-48% acetonitrile; 56-66 min, 48-57% acetonitrile; 66-72 min, 57-70% acetonitrile; 72-78 min, 70-85% acetonitrile; The mass spectrometry conditions were as follows: ion source: electrospray ion source, negative ion mode, data acquisition mode: MSe full scan, range m / z 50-1500.

6. The quality control 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 ; And / or: the C18 chromatographic column in the chromatographic conditions of step ② is Waters CORTECS UPLC C 18 Chromatographic column, 2.1 mm × 100 mm, 1.6 μm, flow rate 0.25 mL min -1 , column temperature 45°C; injection volume 2 μL; The mass spectrometry conditions were: capillary voltage: -3.0 kV, desolvation gas flow: nitrogen, flow rate: 800 L·h -1 , desolvation temperature: 500℃, cone gas flow: nitrogen, cone voltage: 30.0V, flow rate: 50L·h -1 , collision gas: argon, collision energy: 10-55eV, ion source temperature: 120℃.

7. A method for detecting the biological effect of compound Pien Tze Huang buccal 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 the compound Pien Tze Huang lozenges and the RU value of isochlorogenic acid B, wherein the protein chip in the surface plasmon resonance instrument is a TNFR2 protein chip; ② The ratio is calculated using the following formula. If the ratio is not less than 1, the biological effect test of Compound Pien Tze Huang Lozenges is qualified; Ratio = RU value of Compound Pien Tze Huang lozenges / RU value of isochlorogenic acid B.

8. The method according to claim 7, wherein: The steps for determining the RU value of Compound Pien Tze Huang Lozenges and the RU value of isochlorogenic acid B are as follows: The compound Pien Tze Huang lozenge solution and the isochlorogenic acid B reference solution were placed in a surface plasmon resonance test plate loaded with a TNFR2 protein chip. SPR assay conditions were: association time: 60 s; flow rate: 30 μl / min, dissociation time: 60 s; temperature: 25°C; running buffer: 5% DMSO in PBS. The compound Pien Tze Huang buccal tablet test solution is prepared by taking the compound Pien Tze Huang buccal tablet and preparing the fishing test solution according to the method described in step A2; The isochlorogenic acid B reference solution is prepared by dissolving isochlorogenic acid B in DMSO, adding PBS solution to the dissolving solution and mixing the solution with a PBS solution containing DMSO; the concentration of isochlorogenic acid B in the dissolving solution is 10 mM; the volume ratio of the dissolving solution, PBS solution and PBS solution containing DMSO is 5 μl:95 μl:900 μl; The TNFR2 protein chip is constructed by taking TNFR2 protein according to the method for constructing a protein chip described in step A1.

9. A UPLC detection method for active ingredients targeting TNFR2 protein, TLR4 protein and / or TLR5 protein in Pien Tze Huang Compound Lozenges, characterized in that: The method uses UPLC to detect the fingerprint of Compound Pien Tze Huang Lozenges and determine the content of the components in the fingerprint, specifically including the following steps: 1) Preparation of reference solution: Take the reference substance and dissolve it in methanol solution; 2) Preparation of test solution: Take the compound Pien Tze Huang buccal tablets to be tested, extract with methanol solution, filter, and obtain the filtrate; 3) Separately pipette the reference solution and the test solution into a high performance liquid chromatograph; the chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica as filler; mobile phase: formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B; gradient elution program: 0-3 min, 5% B→13% B; 13-16 min, 13% B→15.5% B; 16-25 min, 15.5% B→21% B; 25-28 min, 21% B; 28-32 min, 21% B→22% B; 32-41 min, 22% B→30% B.

10. The UPLC detection method according to claim 9, wherein: Step 1) The concentration of the methanol solution is 50% to 100%; the concentration of each reference substance in the reference solution is 1 to 50 μg·mL -1 ; The reference substances are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid, isoflurane, liquiritin, verbascoside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoside C and / or isoliquiritin. And / or: in step 2), the mass volume ratio of the compound Pien Tze Huang lozenge to be tested and the methanol solution is 1-5 g:10-100 ml; the concentration of the methanol solution is 50%-100%, v / v. And / or: Step 3) The chromatographic conditions are: Chromatographic column: Welch UltimateAQ-C 18 Chromatographic column, 2.1 mm × 150 mm, 1.8 μm, column temperature 30-35 °C, injection volume 2 μL, flow rate 0.25 mg min -1 , detection wavelength 330nm.

11. The UPLC detection method according to claim 9, wherein: The fingerprint of the compound Pien Tze Huang lozenge to be tested should present 18 characteristic peaks, of which 13 peaks should correspond to the retention times of the peaks of the corresponding reference substance; the fingerprint is preferably Figure 7A.

12. The UPLC detection method according to claim 11, wherein: The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, ferulic acid, isoflurane, liquiritin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoside C and / or isoliquiritin in the compound Pien Tze Huang lozenge to be tested are calculated using the peak area according to the external standard method.

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