A quality evaluation method for Angong Niuhuang Wan based on the biological effect of STAT3 protein

Abstract

The present invention relates to a kind of Angong Niuhuang Wan quality evaluation method based on STAT3 protein biological effect, it is to use surface plasmon resonance (SPR) detection technology, specifically including the following steps: 1) construction of protein chip;2) preparation of test solution;3) preparation of reference solution;4) test solution and reference solution injection are taken respectively and carry out SPR detection, analyze and flow through protein chip surface and STAT 3 protein binding, measure corresponding response value (RU value).Quality evaluation standard is that the ratio of test sample RU value to reference substance RU value should be not less than 1.The present invention is optimized and verified by methodology, and accuracy is high, can reflect the effectiveness of Angong Niuhuang Wan clinical treatment, further improves the quality control system of Angong Niuhuang Wan, possesses practical application promotion value.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a quality evaluation method for Angong Niuhuang Pills based on the biological effect of STAT3 protein. Background Art

[0002] Angong Niuhuang Wan is composed of eleven Chinese medicinal materials including bezoar, buffalo horn concentrated powder, musk, pearl, cinnabar, realgar, coptis chinensis, scutellaria baicalensis, gardenia, turmeric, and borneol. It has the effects of clearing away heat and detoxifying, calming the nerves and opening the orifices. It is used for fever, evil entering the pericardium, high fever convulsions, coma and delirium, stroke coma, and encephalitis, meningitis, toxic encephalopathy, cerebral hemorrhage, and sepsis with the above symptoms. The quality standards of Angong Niuhuang Wan in the "Chinese Pharmacopoeia" (2020 edition, Part I) include properties, identification, inspection, content determination and other items. The identification items mainly include microscopic identification of buffalo horn concentrated powder, pearl, cinnabar, realgar, coptis chinensis, scutellaria baicalensis, gardenia, and turmeric, as well as identification methods of bile acid, berberine hydrochloride, borneol, and musk ketone. The content of bilirubin, baicalin, and berberine hydrochloride is used as indicators for content determination. Although the quality standards of Angong Niuhuang Wan cover the quality control of all medicinal ingredients in the prescription, due to the complexity of Chinese medicine ingredients, the material basis of many pharmacological effects has not been fully elucidated. The model of controlling the quality of Chinese medicine with individual indicator chemical components has the problem of being not closely related to clinical effectiveness and safety.

[0003] Chinese invention patent CN109633037B discloses a UPLC fingerprint construction and detection method for Angong Niuhuang Wan. The established UPLC fingerprint calibrated 25 common peaks within 30 minutes and identified 17 of these chromatographic peaks, allowing for more comprehensive and accurate control of the chemical composition of the preparation. However, the fingerprint is easily affected by multiple factors such as the medicinal material variety, origin, planting conditions, harvest period, processing method, and experimental conditions. In addition, there is a lack of research on the correlation between fingerprints and pharmacological effects, which limits its practical application.

[0004] There are currently no reports on the use of bioeffect evaluation methods for quality control of Angong Niuhuang Wan. Summary of the Invention

[0005] The purpose of the present invention is to provide a biological effect detection method for reflecting and evaluating the pharmacological effects of Angong Niuhuang Pills, so as to make up for the problem that the existing quality standards of Angong Niuhuang Pills are not closely related to clinical effectiveness and safety, and further improve the quality control system of the product.

[0006] The present invention also provides a quality evaluation method for Angong Niuhuang Wan based on the biological effect of STAT3 protein, which adopts SPR detection and specifically comprises the following steps:

[0007] 1) Construction of protein chip: Dissolve STAT3 protein in water, then dilute with sodium acetate buffer to a 10-40 μg / ml protein solution. The protein solution is coupled to the sensor chip.

[0008] 2) Preparation of test solution: Take Angong Niuhuang Wan, extract with 80% methanol solution, centrifuge the extract, dry the supernatant, re-dissolve with DMSO, filter, and mix the filtrate with PBS solution.

[0009] 3) Preparation of reference solution: Dissolve berberine in DMSO, add PBS solution and DMSO-containing PBS solution to the dissolved solution and mix well.

[0010] 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);

[0011] SPR detection conditions: run time: 60 s; flow rate: 30 μl / min, dissociation time: 60 s, temperature: 25° C. Running buffer: 5% DMSO in PBS.

[0012] Furthermore, the protein solution in step 1) is 20 μg / ml.

[0013] Furthermore, the coupling in step 1) is amino coupling; the parameters of the amino coupling are sensor chip model: CM7 or CM5, contact time: 420s, flow rate: 10μl / min, temperature: 25°C, coupling reagents: EDC and NHS, blocking reagent ethanolamine, running buffer: 1×HBS-EP.

[0014] Furthermore, the chip model is CM7, the volume ratio of the protein solution, EDC and NHS, and ethanolamine is 100:200:140; and the volume ratio of EDC and NHS is 1:1.

[0015] Furthermore, in step 2), the mass volume ratio of the Angong Niuhuang Pills to the 80% methanol solution is 0.1 g: 5-25 ml; the extraction is ultrasonic extraction, the time is 20-60 min, the power is 20-80 kHz, 100-500 W; the centrifugal speed is 5000-25000 r, the time is 5-25 min; the amount of DMSO added is 15-25 times that of the supernatant.

[0016] Furthermore, in step 3), the concentration of berberine in the dissolving solution is 10 mM; and the volume ratio of the dissolving solution, PBS solution and PBS solution containing DMSO is 5 μl:95 μl:900 μl.

[0017] Furthermore, the DMSO concentration in the PBS solution containing DMSO is 5%.

[0018] Furthermore, the ratio of the RU value of Angong Niuhuang Wan detected by SPR to the RU value of berberine is not less than 1.

[0019] The present invention also provides an application of STAT3 protein as a biological quality marker in the quality detection of Angong Niuhuang Pills.

[0020] Furthermore, the detection is to detect the biological effect of Angong Niuhuang Wan using surface plasmon resonance method.

[0021] The present invention constructs a PPI network of Angong Niuhuang Pills' component targets and stroke disease targets, screens potential targets of Angong Niuhuang Pills for the clinical treatment of stroke, establishes a bioquality marker screening model, and ultimately screens out the STAT3 target protein as the optimal bioquality marker of Angong Niuhuang Pills.

[0022] The present invention adopts STAT3 protein fishing chip to fish the chemical components of Angong Niuhuang Pills, and obtains 10 active compounds through biological quality marker activity verification and screening. After methodological optimization and verification, a STAT3-based biological effect detection method of Angong Niuhuang Pills is established. The method is simple, easy to operate, and highly accurate. It can reflect the effectiveness of the clinical treatment of Angong Niuhuang Pills, further improve the quality control system of Angong Niuhuang Pills, and has practical application and promotion value.

[0023] 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.

[0024] The following further describes the above contents of the present invention in detail through specific implementation methods in the form of examples, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Pre-enrichment results of 5 proteins on chip

[0026] Figure 2 SPR pre-screening results of Angong Niuhuang Wan on STAT3 protein

[0027] Figure 3 SPR pre-screening results of Angong Niuhuang Wan on DKK-1 protein

[0028] Figure 4 LC-Q-TOF-MS images of fishing components

[0029] Figure 5Pre-enrichment results of three protein concentrations

[0030] Figure 6 Berberine concentration selection

[0031] Figure 7 STAT3 pre-enrichment results on CM7

[0032] Figure 8 STAT3 pre-enrichment results on NTA

[0033] Figure 9 STAT3 pre-enrichment results on CM5 DETAILED DESCRIPTION

[0034] Example 1 Quality inspection of Angong Niuhuang Pills of the present invention

[0035] 1) Construction of protein chip:

[0036] Dissolve STAT3 protein in sterile water to obtain a 500 μg / ml protein stock solution. Take 8 μl of the protein stock solution and add 192 μl of 10 mM pH 5.0 sodium acetate buffer and mix well to obtain a 20 μg / ml protein solution.

[0037] The surface plasmon resonance instrument was started, with channel 1 set as the blank unit and channel 2 as the coupling unit. 100 μl of the protein solution was taken and amino-coupled to the CM7 chip. The coupling parameters were: contact time: 420 s, flow rate: 10 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), and 140 μl of ethanolamine (from the amino coupling kit) was used for blocking. Running buffer: 1×HBS-EP.

[0038] 2) Preparation of test solution:

[0039] Take 0.1g of Angong Niuhuang Wan pills and place them in a 25ml conical flask. Add 10ml of 80% methanol and extract by ultrasonication for 30min (50kHz, 350W). Make up the weight loss with 80% methanol. Centrifuge at 14000r for 10min. Accurately aspirate 2ml of the supernatant, concentrate by centrifugation at 4℃ and evaporate to dryness. Add 200μl of DMSO to redissolve the supernatant. Pass the supernatant through a 0.22μm pore filter. Accurately aspirate 10μl of the supernatant and add 190μl of PBS 1× solution. Mix well.

[0040] 3) Preparation of reference solution:

[0041] Accurately weigh berberine and prepare it into a stock solution with DMSO to a concentration of 10 mM. Take 5 μl of the control stock solution, add 95 μl of PBS 1× and 900 μl of PBS solution containing 5% DMSO and mix well.

[0042] 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);

[0043] 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.

[0044] The beneficial effects of the present invention are further illustrated by the following test examples:

[0045] Test Example 1

[0046] 1. Screening of biological quality markers (protein targets)

[0047] 1. Experimental instruments and test drugs

[0048] Biacore T200 (GE, USA), Milli-Q ultrapure water instrument (Millipore, USA) Proteins: STAT3 (Abcam, UK, ab268982), DKK-1 (Abcam, UK, ab155623), VEGFR1 (Abcam, UK, ab84771), TRPM7 (Abcam, UK, batch number ab153385), PKM2 (Abcam, UK, ab153385)

[0049] Buffers: HBS-EP + 10× buffer solution (GE, USA, BR-1006-69), PBS 10× buffer solution (GE, USA, BR-1006-72), DMSO (VETEC, Germany, V900090), CM5 sensor chip (GE, USA, BR-1005-30), sterile water was MillQ ultrapure water sterilized by autoclave; amino coupling kit (GE, USA, BR-1000-50), sodium acetate pH 4.5 (GE, USA, BR-1003-50), sodium acetate pH 5.0 (GE, USA, BR-1003-51), sodium acetate pH 5.5 (GE, USA, BR-1003-52), NaOH 50 (GE, USA, BR-1003-58), positive drug tripterygium wilfordii (Chengdu Mansite Biotechnology Co., Ltd. A0106), negative drug aliertib (MLN 8237) were purchased from Med Chem. Express Company, Angong Niuhuang Pills (Longhui Pharmaceutical Co., Ltd., batch numbers: 2021004, 2021005, 2021006).

[0050] 2. Experimental Methods and Results

[0051] 2.1 Solution preparation

[0052] 2.1.1 Preparation of protein solution

[0053] STAT3, DKK, VEGFR1, TRPM7, and PKM2 proteins were precisely aspirated and diluted with sterile water to a 500 μg / ml protein stock solution for protein chip construction.

[0054] 2.1.2 Preparation of buffer for chip construction

[0055] The buffer used in this experiment is HBS-EP. Dilute 10×HBS-EP 10 times to prepare 500 ml of running buffer.

[0056] 2.1.3 Preparation of Biacore protein chip screening running buffer

[0057] PBS 1× buffer was diluted 10-fold with ultrapure water to obtain PBS 1× buffer. DMSO was added to PBS 1× buffer to prepare a 5% (ml / ml) DMSO-containing PBS solution, which was used as the running buffer for Biacore protein chip screening.

[0058] 2.1.4 Preparation of test samples

[0059] Preparation of sample solution: Take 0.1g of Angong Niuhuang Wan pills, place them in a 25ml conical flask, add 10ml of 80% methanol, ultrasonically extract for 30min (50kHz, 350W), make up the weight loss with 80% methanol, centrifuge at 14000r for 10min, accurately aspirate 2ml of supernatant, centrifuge and concentrate to dryness, add 200μl DMSO to re-dissolve, filter through 0.22μm pore membrane, accurately aspirate 10μl, add 190μl PBS 1× solution, mix well.

[0060] Preparation of the positive drug, tripterine: Accurately weigh an appropriate amount of tripterine and prepare a stock solution with DMSO to a concentration of 10 mM. Take 5 μl of the control stock solution, add 95 μl of 1× PBS and 900 μl of 5% DMSO in PBS, and mix thoroughly to a solution concentration of 50 μM in 5% DMSO in PBS. The negative drug, aliertib, was prepared in the same manner.

[0061] 2.2 Protein chip construction

[0062] 2.2.1 Protein pre-enrichment

[0063] 1) Take 4 μl of ligand (5 protein solutions) and add them to 96 μl of pH 5.5 sodium acetate buffer respectively and mix thoroughly. The final concentration of the ligand is about 20 μg / ml.

[0064] 2) Protein preconcentration: Flow rate: 10 μl / min; Flow path: Flow path 2, select Sample and Reagent Rack 1 mode for preconcentration; Contact time: 120 s.

[0065] 3) Pre-enrichment results are shown in Figure 1 TRPM7 and PKM2 could not be enriched with the CM5 chip, and the protein coupling amount of VEGFR1 was too low (target protein fixed amount RU value = target protein molecular weight × analyte RU value / analyte molecular weight, analyte RU value was set to 100RU), failing to achieve the expected enrichment amount (generally above 10,000). STAT3 and DKK-1 could be coupled to the CM5 chip and achieved the expected enrichment amount (above 10,000). Therefore, based on the pre-enrichment results of the five proteins, STAT3 and DKK-1 were selected for further coupling investigation.

[0066] 2.2 SPR pre-screening of STAT3 and DKK-1 proteins by Angong Niuhuang Wan

[0067] 100 μl of each of the Angong Niuhuang Wan sample, the positive drug tripterygium wilfordii, and the negative drug aliertib test solution were precisely pipetted into a 96-well Biacore test plate. The sample was run on the chip with a contact time of 60 s, a flow rate of 30 μl / min, a dissociation time of 60 s, and a temperature of 25°C. Running buffer: 5% DMSO in PBS. The results of the above sample solutions and the STAT3 protein and DDK-1 assays are shown in the table. Figure 2 and Figure 3 As can be seen from the figure, on the STAT3 protein chip, the Angong Niuhuang Wan extract sample had a good response signal, which was better than the positive tripterygium wilfordii, while the negative and solvent in the chip model showed blanks; however, for the DKK-1 protein chip, although the positive (using DKK-1 neutralizing antibody, 20μg / ml) had a response signal, the negative and solvent both showed blank signals, and the response value of Angong Niuhuang Wan was much lower than the response value of Angong Niuhuang Wan on STAT3. Therefore, after the initial screening of protein targets, STAT3 protein was determined as the target for further research.

[0068] 3. Discussion

[0069] According to the clinical efficacy positioning of Angong Niuhuang Wan, the functionally related target proteins STAT3 (regulating Th17 / Treg), DKK-1 (regulating Wnt pathway), VEGF receptor (regulating VEGF pathway), TRPM ion channel (regulating ion channel), and PKM2 / MMP (regulating blood-brain barrier permeability) were selected. By preliminarily constructing a Q-biomarker screening model, the results showed that the protein STAT3 had a good response as a target and could be used as a protein for further bio-quality marker fishing. Therefore, further fishing research was carried out using the STAT3 protein chip.

[0070] 2. Target Q-biomarker Detection and Component Identification of Angong Niuhuang Wan

[0071] A protein fishing chip for the biomarker STAT3 was constructed to screen the chemical components of Angong Niuhuang Wan. The components were identified by LC-MS and compared with reference substances.

[0072] 1. Instruments and test drugs

[0073] Instruments: Biacore T200 (GE, USA), ultrahigh pressure infusion system (model I-class plus) connected in series with Zevo XS high-resolution time-of-flight mass spectrometer (Waters, USA), CPA225D 1 / 100,000 analytical balance (Sartorius, USA), Milli-Q ultrapure water analyzer (Millipore, USA).

[0074] Protein: STAT3 (abcam, UK, ab268982).

[0075] Buffer: HBS-EP+10× buffer solution (GE, USA, BR-1006-69), PBS 10× buffer solution (GE, USA, BR-1006-72), DMSO (VETEC, Germany, V900090).

[0076] Sterile water was MillQ ultrapure water, which was used after high-pressure sterilization.

[0077] CM5 sensor chips (GE, USA, BR-1005-30), amino coupling kit (GE, USA, BR-1000-50), sodium acetate pH 4.5 (GE, USA, BR-1003-50), sodium acetate pH 5.0 (GE, USA, BR-1003-51), sodium acetate pH 5.5 (GE, USA, BR-1003-52), NaOH 50 (GE, USA, BR-1003-58), and 14 compounds used for qualitative identification were purchased from Chengdu Mansite Biotechnology Co., Ltd. Angong Niuhuang Wan (Longhui Pharmaceutical Co., Ltd., batch numbers: 2021004, 2021005, 2021006), methanol, acetonitrile (mass spectrometry grade, Merck, Germany), and formic acid (chromatographic grade, batch number F190210, Aladdin Reagent Shanghai Co., Ltd.) were of analytical grade.

[0078] 2 Experimental methods and results

[0079] 2.1 Solution preparation

[0080] 2.1.1 Preparation of buffer for chip construction

[0081] The buffer used in this experiment is HBS-EP. Dilute 10×HBS-EP 10 times to prepare 500 ml of running buffer.

[0082] 2.1.2 Preparation of test samples

[0083] Preparation of fishing test samples: take 0.1g of Angong Niuhuang Wan pills, place it in a 25ml conical flask, add 10ml of 80% methanol, ultrasonically extract for 30min (50kHz, 350W), make up for the weight loss with 80% methanol, centrifuge at 14000r for 10min, accurately aspirate 2ml of supernatant, centrifuge and concentrate to dryness, add 200μl DMSO to dissolve, filter through 0.22μm pore membrane, accurately aspirate 10μl, add 190μl PBS 1× solution, mix well.

[0084] Preparation of reference solution: Accurately weigh appropriate amounts of coptisine, palmatine, tetrahydrocoptisine, epiberberine, methylcoptisine, demethyleneberberine, berberine, jatrorrhizine, baicalein, baicalin methyl ester, and isoquercetin reference substances, dilute them with pure methanol to a reference substance stock solution of 1.0 mg / ml, and then dilute with 50% methanol to about 1 μg / ml for qualitative identification.

[0085] 2.2 Ligand coupling

[0086] Accurately pipette 16 μl of protein stock solution and add 384 μl of 10 mM pH 5.0 sodium acetate buffer solution. Mix well for coupling. Coupling parameters: Chip type: CM5, Flow cells per cycle: 4 (for fishing, all 4 channel units are coupled, 100 μl of protein solution per channel unit), method: amine coupling, ligand: STAT3 protein, coupling mode: specify contact time and flow rate, contact time: 420 s, flow rate: 10 μl / min. Temperature: 25°C. Coupling reagents: 100 μl each of EDC and NHS (from the amino coupling kit), and 140 μl of ethanolamine (from the amino coupling kit) is used for blocking.

[0087] 2.3 Fishing and Retrieving

[0088] Fishing and recovery experimental parameters: contact time: 180 s, flow rate 5 μl / min, incubation time: 20 s, wash solution: ultrapure water, recovery solution: 0.5% TFA, deposition solution: 50 mM NH4HCO3 (used as a collection solution to dissociate active ingredients bound to the chip surface). Cycles: 7, number of replicates: 17 times.

[0089] Control group experiment: Use a new CM5 chip without ligand coupling, and perform injection and recovery directly on the blank CM5 chip. The experimental operation and parameters of fishing are the same as those of the experimental group. The recovered samples are used as negative control for LC-MS detection.

[0090] UPLC-MS analysis

[0091] 2.4.1 Chromatographic conditions

[0092] CORTECS™ UPLC C18 (2.1×100 mm, 1.6 μm), mobile phase acetonitrile (B)-0.1% formic acid water (A), gradient elution (0-0.5 min, 12-12% B; 0.5-2.0 min, 12-25% B; 2.0-4.0 min, 25-32% B;

[0093] 4.0-6.0min, 32%B; 6.0-10.0min, 32-65%B; 10.0-12.0min 65%B, volume flow rate 0.25ml / min, column temperature 45℃, injection volume 2μl.

[0094] 2.4.2 Mass spectrometry conditions

[0095] Zevo XS high-resolution time-of-flight mass spectrometer, using electrospray ionization (ESI) in positive and negative ion modes, with a positive ion capillary voltage of 3.0 kV and a negative ion capillary voltage of 2.5 kV, was used in MS1 ​​Scan mode with a mass scan range of m / z 50 to 2000, a desolvation gas flow of N2 at a flow rate of 800 L·h-1, a desolvation temperature of 400°C, a cone gas flow of N2 at a flow rate of 50 L·h-1, and an ion source temperature of 120°C. Under these chromatographic and mass spectrometric conditions, the total ion current chromatogram obtained is shown in Figure 2. Figure 4 .

[0096] 2.5 Mass spectrometry results

[0097] A total of 10 chemical components were identified based on the total ion current of UPLC-Q-TOF-MS of Angong Niuhuang Wan. The specific identification method was as follows: first, based on the accurate mass number obtained from the total ion current chromatographic peak of high-resolution mass spectrometry, the molecular ion peak information (MS1) of each chromatographic peak was obtained. Then, the exact molecular formula was calculated within a mass deviation of 10 ppm using the Tools Elemental Composition software in Masslynx 4.2 software. The components were assigned to the single herb, and the chemical components that had been preliminarily identified were compared with reference substances purchased to accurately identify each chromatographic peak. Finally, a total of 10 compounds were identified through comparison with reference substances: methylberberine, desmethyleneberberine, tetrahydroberberine, palmatine, epiberberine, jatrorrhizine, coptisine, berberine, baicalein, and baicalin methyl ester.

[0098] 3. Verification of Q-biomarker affinity activity of fishing ingredients

[0099] The effects of different concentrations of the screened components on the Q-biomarker activity were studied, and affinity kinetic parameters were calculated to verify the affinity activity.

[0100] 1. Instruments and test drugs

[0101] Instruments: Biacore T200 (GE, USA), ultrahigh pressure infusion system (model I-class plus) connected in series with Zevo XS high-resolution time-of-flight mass spectrometer (Waters, USA), CPA225D 1 / 100,000 analytical balance (Sartorius, USA), Milli-Q ultrapure water analyzer (Millipore, USA).

[0102] Protein: STAT3 (abcam, UK, ab268982).

[0103] Buffer: HBS-EP + 10× buffer solution (GE, USA, BR-1006-69), PBS 10× buffer solution (GE, USA, BR-1006-72), DMSO (VETEC, Germany, V900090). Sterile water was MillQ ultrapure water, which was sterilized by autoclave before use.

[0104] CM5 sensor chips (GE, USA, BR-1005-30), amino coupling kit (GE, USA, BR-1000-50), sodium acetate pH 4.5 (GE, USA, BR-1003-50), sodium acetate pH 5.0 (GE, USA, BR-1003-51), sodium acetate pH 5.5 (GE, USA, BR-1003-52), NaOH 50 (GE, USA, BR-1003-58), and 14 compounds used for qualitative identification were purchased from Chengdu Mansite Biotechnology Co., Ltd. Angong Niuhuang Wan (Longhui Pharmaceutical Co., Ltd., batch numbers: 2021004, 2021005, 2021006), methanol, acetonitrile (mass spectrometry grade, Merck, Germany), and formic acid (chromatographic grade, batch number F190210, Aladdin Reagent Shanghai Co., Ltd.) were of analytical grade.

[0105] 2. Experimental Methods and Results

[0106] 2.1 Solution preparation

[0107] 2.1.1 Configuration of chip construction buffer

[0108] The buffer used in this experiment is HBS-EP. Dilute 10×HBS-EP 10 times with ultrapure water to obtain 500 ml of 1×HBS-EP, and prepare 500 ml of protein pre-enrichment and coupling running buffer.

[0109] 2.1.2 Preparation of running buffer for SPR screening and affinity analysis

[0110] PBS 1× buffer was diluted 10-fold with ultrapure water to obtain PBS 1× buffer. DMSO was added to PBS 1× buffer to prepare a 5% (ml / ml) DMSO-containing PBS solution, which was used as the running buffer for Biacore protein chip screening.

[0111] 2.1.3 Preparation of test samples

[0112] Preparation of test samples: Accurately weigh appropriate amounts of 69 reference substances, prepare a stock solution concentration of 10 mM with DMSO, take 5 μl of the reference substance stock solution, add 95 μl PBS 1× and 900 μl of PBS solution containing 5% DMSO, and mix to prepare a solution concentration of 50 μM PBS solution containing 5% DMSO.

[0113] Preparation of the positive drug, tripterine: Accurately weigh an appropriate amount of tripterine and prepare a stock solution with DMSO to a concentration of 10 mM. Take 5 μl of the control stock solution, add 95 μl of 1× PBS and 900 μl of 5% DMSO in PBS, and mix thoroughly to a solution concentration of 50 μM in 5% DMSO in PBS. The negative drug, aliertib, was prepared in the same manner.

[0114] 2.1.4 Preparation of solvent calibration solution

[0115] According to Table 1, PBS 1× solutions containing 4.5% DMSO and 5.8% DMSO were prepared, respectively.

[0116] Table 1 Preparation of solvent calibration solution

[0117]

[0118] 2.2 SPR detection of STAT3 by identification components

[0119] 10 fishing components (coptisine, palmatine, tetrahydrocoptisine, epiberberine, methylcoptisine, demethyleneberberine, berberine, jatrorrhizine, baicalein, baicalin methyl ester) detected and identified, as well as the test solutions of the positive drug tripterygium wilfordii and the negative drug Aliertib, as well as the blank solvent, were accurately aspirated and placed in a 96-well Biacore test plate. The sample was tested in the chip with the following running time: contact time: 60s; Flow rat: 30μl / min, Dissociation time: 60s, temperature: 25℃. Running Buffer: 5% DMSO in PBS) and the corresponding test tube rack was replaced, and the position of the EP tube was adjusted according to the operation panel. Finally, 10 components with positive results were screened out: coptisine, palmatine, tetrahydrocoptisine, epiberberine, methylcoptisine, demethyleneberberine, berberine, jatrorrhizine, baicalein, and baicalin methyl ester.

[0120] 2.3 SPR affinity KD analysis of 10 fishing components

[0121] The ten selected components were subjected to affinity analysis. Test solutions in PBS containing 5% DMSO were prepared at the following concentrations: 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.7813 μM, 0.3906 μM, 0.1953 μM, 0.0977 μM, 0.0488 μM, and 0 μM.

[0122] Affinity analysis parameters were set as follows: Flow path: 2-1, Chip type: CM5, contact time: 180 s, Flow rate: 30 μl / min, Dissociation time: 300 s, and temperature: 25°C.

[0123] The experimental results are shown in Table 2.

[0124] Table 2 KD values ​​(Mol) of affinity of ten active ingredients

[0125]

[0126] 3. Discussion

[0127] According to the results in the table, the KD values ​​of the ten components are of the same order of magnitude. Among them, berberine is selected as the positive control for the construction of the bioanalysis method, and chlorogenic acid is selected as the negative control because of its high content in Angong Niuhuang Wan and its inclusion in the pharmacopoeia.

[0128] 4. Development and Validation of Q-biomarker Methodology

[0129] 1. Experimental instruments and test drugs

[0130] Instruments: Biacore T200 (GE, USA), CPA225D 1 / 100,000 analytical balance (Sartorius, USA), Milli-Q ultrapure water analyzer (Millipore, USA).

[0131] Protein: STAT3 (abcam, UK, ab268982)

[0132] Buffer: HBS-EP + 10× buffer solution (GE, USA, BR-1006-69), PBS 10× buffer solution (GE, USA, BR-1006-72), DMSO (VETEC, Germany, V900090)

[0133] Sterile water was MillQ ultrapure water, which was used after high-pressure sterilization;

[0134] CM5 sensor chip (GE, USA, BR-1005-30), amino coupling kit (GE, USA, BR-1000-50), sodium acetate pH 4.5 (GE, USA, BR-1003-50), sodium acetate pH 5.0 (GE, USA, BR-1003-51), sodium acetate pH 5.5 (GE, USA, BR-1003-52), regeneration kit (GE, USA, BR-1005-56), NaOH 50 (GE, USA, BR-1003-58), positive drug berberine (Chengdu Mansite Biotechnology Co., Ltd. A0152), negative drug chlorogenic acid (Chengdu Mansite Biotechnology Co., Ltd. A0022), Angong Niuhuang Pills (Longhui Pharmaceutical Co., Ltd., batch numbers: 2021004, 2021005, 2021006).

[0135] 2. Experimental Methods and Results

[0136] 2.1 Specificity Experiment

[0137] In order to verify that Angong Niuhuang Wan did not bind to STAT3 non-specifically, IGF-Ⅰ, IGF-Ⅱ and blank chips were used for verification.

[0138] 2.1.1 Preparation of test samples

[0139] Preparation of sample solution: Take 0.1g of Angong Niuhuang Wan pills, place them in a 25ml conical flask, add 10ml of 80% methanol, ultrasonically extract for 30min (50kHz, 350W), make up the weight loss with 80% methanol, centrifuge at 14000r for 10min, accurately aspirate 2ml of supernatant, centrifuge and concentrate to dryness, add 200μl DMSO to re-dissolve, filter through 0.22μm pore membrane, accurately aspirate 10μl, add 190μl PBS 1× solution, mix well.

[0140] Preparation of the positive agent berberine: Accurately weigh an appropriate amount of berberine 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 1x PBS and 900 μl of 5% DMSO in PBS, and mix thoroughly to a 50 μM solution in 5% DMSO in PBS. Chlorogenic acid, a negative agent, is prepared in the same manner.

[0141] 2.1.2 Protein chip construction

[0142] The STAT3 chip was constructed as in the previous experiment, and the IGF-I and IGF-II chips were used for methodological specificity studies. A chip without coupled protein was used as a blank control.

[0143] 2.1.3 SPR detection of STAT3, IGF-I and IGF-II proteins by Angong Niuhuang Wan

[0144] 100 μl of each of the Angong Niuhuang Wan sample, the positive drug berberine, and the negative drug chlorogenic acid test solution was precisely pipetted into a 96-well Biacore assay plate. The samples were run during the chip screening process with a contact time of 60 seconds, a flow rate of 30 μl / min, a dissociation time of 60 seconds, and a temperature of 25°C. The running buffer consisted of 5% DMSO in PBS. The RU values ​​for the four groups are shown in Table 3.

[0145] The results show that on the STAT3 protein chip, the Angong Niuhuang Wan extract sample has a good response signal with it, which is better than the positive berberine, while the negative and solvent both show blank; the detection results of the IGF-Ⅰ and IGF-Ⅱ chips are all negative (similarly, the positive drug berberine has no detection signal in IGF-Ⅰ and IGF-Ⅱ, showing a negative result). In addition, the blank chip (without protein coupling) also has a negative result. Therefore, through specific experiments, it can be verified that Angong Niuhuang Wan has specific binding to STAT3.

[0146] Table 3 Detection results of Angong Niuhuang Wan on STAT3 chip, IGF-Ⅰ chip, IGF-Ⅱ chip and blank chip

[0147]

[0148] 2.2 Precision, repeatability and stability tests

[0149] 2.3.1 Precision test

[0150] Angong Niuhuang Wan sample (batch number 2021005) was prepared using the method in 2.3.1. Six 100 μl aliquots were precisely pipetted into a 96-well Biacore plate. The sample was screened using a 60-second contact time, a 30 μl / min flow rate, a 60-second dissociation time, and a temperature of 25°C. The running buffer used was 5% DMSO in PBS. The RU values ​​for the six screening runs were 842.9, 839.3, 839.3, 841.1, 831.7, and 874.3, respectively, with an RSD of 1.8%, indicating good instrument precision. Berberine was screened six times using the same method, yielding RU values ​​of 36.5, 36.9, 37.8, 38.4, 37.4, and 35.3, respectively, with an RSD of 2.9%.

[0151] 2.3.2 Repeatability test

[0152] Six samples of Angong Niuhuang Wan (lot 2021005) were collected from the same batch and the test solution was prepared according to the method in 2.3.1. 100 μl was precisely pipetted and placed into a 96-well Biacore plate. The sample was screened using a contact time of 60 s, a flow rate of 30 μl / min, a dissociation time of 60 s, and a temperature of 25°C. The running buffer used was 5% DMSO in PBS. The RU values ​​for the six screening runs were 792.7, 869.4, 912.6, 813.2, 920.6, and 837.9, respectively. The RSD of the RU values ​​was 6.1%, indicating good reproducibility of the method.

[0153] 2.3.3 Sample stability test

[0154] Angong Niuhuang Wan sample (batch number 2021005) was prepared according to the method in 2.2.1. 100 μl of the sample was precisely pipetted into a 96-well Biacore plate. SPR screening was performed at 0, 2, 4, 8, 12, and 24 h. The sample was screened using a contact time of 60 s, a flow rate of 30 μl / min, a dissociation time of 60 s, and a temperature of 25°C. Running buffer: 5% DMSO in PBS. The RU values ​​for the six screening runs were 792.7, 779, 784.2, 698.2, 829.3, and 839.3, respectively. The RSD of the RU values ​​was 6.3%, indicating that the sample solution was stable at room temperature for 24 h.

[0155] 2.4 Durability and Optimization of Protein Concentration

[0156] 1) 10 μg / ml protein solution: 2 μl protein stock solution + 98 μl sodium acetate buffer of different pH values;

[0157] 20 μg / ml protein solution: 4 μl protein stock solution + 96 μl sodium acetate buffer of different pH values;

[0158] 40μg / ml protein solution: 8μl protein stock solution + 92μl sodium acetate buffer of different pH values

[0159] 2) Protein preconcentration: Flow rate: 10 μl / min; Flow path: Flow path 2, select Sample and Reagent Rack 1 mode for preconcentration; Contact time: 120 s. Preconcentrate at three protein concentrations.

[0160] 3) Pre-enrichment results are shown in Figure 5As can be seen from the figure, the coupling amount at a concentration of 10 μg / ml is significantly lower than the other two concentrations, while the coupling amount at a concentration of 40 μg / ml is not much different from the coupling amount at a concentration of 20 μg / ml. It is possible that the chip has reached saturation and cannot couple more proteins, so the 20 μg / ml protein solution was finally selected as the coupling concentration.

[0161] 2.4 Selection of reference substance concentration

[0162] 2.4.1 Preparation of berberine solution

[0163] The test sample solutions in PBS containing 5% DMSO were prepared in the following concentrations: 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.7813 μM, 0.3906 μM, 0.1953 μM, 0.0977 μM, 0.0488 μM and 0 μM.

[0164] 2.4.2 Affinity screening of berberine

[0165] Affinity analysis parameters were set as follows: Flow path: 2-1, Chip type: CM5, contact time: 180 s, Flow rate: 30 μl / min, Dissociation time: 300 s, and temperature: 25°C.

[0166] The experimental results are shown in Figure 6 , the KD value is 5.275×10 -5 Mol, and finally 50 μM was selected as the concentration of the control.

[0167] 2.5 Optimization of test products

[0168] 2.5.1 Preparation of test samples

[0169] Take 12 portions of 0.1g pills respectively, place them in 12 25ml conical flasks, add 20%, 40%, 60%, 75%, 80%, 100% methanol and 10ml of ethanol respectively, extract by ultrasonication for 30min (50kHz, 350W), make up the weight loss with the corresponding solvent, centrifuge at 14000r for 10min, filter through 0.22μm pore membrane, and dilute 25 times with 50% methanol to obtain the test solution.

[0170] 2.5.2 Chromatographic conditions

[0171] CORTECS™ UPLC C18 (2.1×100 mm, 1.6 μm), mobile phase: acetonitrile (B)-0.1% formic acid in water (A), gradient elution (0-0.5 min, 12-12% B; 0.5-2.0 min, 12-25% B; 2.0-4.0 min, 25-32% B; 4.0-6.0 min, 32% B; 6.0-10.0 min, 32-65% B; 10.0-12.0 min 65% B, flow rate 0.25 ml / min, column temperature 45°C, injection volume 2 μl.

[0172] 2.5.3 Mass spectrometry conditions

[0173] Zevo XS high-resolution time-of-flight mass spectrometer was used for detection in positive and negative ion modes using an electrospray ionization (ESI) source with a positive ion capillary voltage of 3.0 kV and a negative ion capillary voltage of 2.5 kV. The MS1 Scan mode was used with a mass scan range of m / z 50 to 2000. The desolvation gas flow was N2 at a flow rate of 800 L·h-1, the desolvation temperature was 400°C, and the cone gas flow was N2 at a flow rate of 50 L·h-1. -1 , ion source temperature 120 ° C. Under the chromatographic and mass spectrometric conditions, the total ion current chromatogram was collected, and the pharmacopoeial index ingredient baicalin was used as a reference.

[0174] 2.5.4 Results

[0175] According to the UPLC-Q-TOF-MS total ion current results of Angong Niuhuang Wan extracted with different solvents, it was found that the effect of Angong Niuhuang Wan extracted with 80% methanol was better than that of Angong Niuhuang Wan extracted with other solvents.

[0176] 2.6 Durability and Optimization of DMSO Amount

[0177] 2.6.1 Preparation of test samples

[0178] Preparation of sample solution: Take 0.1g of pills, place them in a 25ml Erlenmeyer flask, add 10ml of 80% methanol, ultrasonically extract for 30min (50kHz, 350W), make up the weight loss with 80% methanol, centrifuge at 14000r for 10min, accurately aspirate 2ml, centrifuge and concentrate to dryness, add different volumes (100, 200, 400, 1000μl) of DMSO to reconstitute (100 reconstitution ratio is 1:5, 200 reconstitution ratio is 1:10, 400 reconstitution ratio is 1:20, 1000 reconstitution ratio is 1:50), filter through 0.22μm pore membrane to obtain test samples with different reconstitution ratios.

[0179] Preparation of the positive agent berberine: Accurately weigh an appropriate amount of berberine 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 PBS and 900 μl of a 5% DMSO-containing PBS solution, and mix thoroughly to a 50 μM solution in 5% DMSO-containing PBS. Chlorogenic acid, a negative agent, is prepared in the same manner.

[0180] 2.6.2 Screening of STAT3 by Angong Niuhuang Wan at different reconstitution ratios

[0181] 100 μl of each Angong Niuhuang Wan sample, the positive drug berberine, and the negative test solution were precisely pipetted into a 96-well Biacore plate. The sample was run under a contact time of 60 seconds, a flow rate of 30 μl / min, a dissociation time of 60 seconds, and a temperature of 25°C. The running buffer consisted of 5% DMSO in PBS. The results indicated that saturation was reached with a volume above 200 μl, so a 200 μl volume was used for reconstitution.

[0182] 2.7 Chip Durability and Optimization

[0183] The CM5 sensor chip is a general-purpose chip, the CM7 sensor chip is used when a higher coupling level is required when studying small molecules, and the NTA sensor chip is suitable for coupling molecules with a His tag.

[0184] 2.7.1 Preparation of test samples

[0185] Preparation of sample solution: Take 0.1g of Angong Niuhuang Wan pills, place them in a 25ml conical flask, add 10ml of 80% methanol, ultrasonically extract for 30min (50kHz, 350W), make up the weight loss with 80% methanol, centrifuge at 14000r for 10min, accurately aspirate 2ml of supernatant, centrifuge and concentrate to dryness, add 200μl DMSO to re-dissolve, filter through 0.22μm pore membrane, accurately aspirate 10μl, add 190μl PBS solution, mix well.

[0186] Preparation of the positive drug berberine: Accurately weigh an appropriate amount of berberine and prepare a stock solution with DMSO to a concentration of 10 mM. Take 5 μl of the control stock solution, add 95 μl of PBS and 900 μl of 5% DMSO in PBS, and mix thoroughly to a solution concentration of 50 μM in 5% DMSO in PBS. Prepare the negative drug in the same manner.

[0187] 2.7.2 Chip Construction

[0188] 2.7.2.1 Protein pre-enrichment

[0189] 1) Take 4 μl of ligand (5 protein solutions) and add them to 96 μl of pH 5.5 sodium acetate buffer respectively and mix thoroughly. The final concentration of the ligand is about 20 μg / ml.

[0190] 2) Protein preconcentration: Flow rate: 10 μl / min; Flow path: Flow path 2, select Sample and Reagent Rack 1 mode for preconcentration; Contact time: 120 s.

[0191] 3) CM7 pre-enrichment results are shown in Figure 7 , NTA pre-enrichment results are shown in Figure 8 , the pre-enrichment results of CM5 are shown in Figure 9 .

[0192] 2.7.2.2 Protein Coupling

[0193] Accurately pipette 8 μl of protein stock solution (STAT3) and add 192 μl of 10 mM sodium acetate buffer (pH 5.0). Mix thoroughly for coupling. Take 100 μl of the protein solution and set the coupling parameters to Chip type: CM7, Flow cells PE: 2 (coupling channel set to the second channel unit), Method: Amine coupling, Ligand: STAT3 protein, Coupling mode: Specify contact time and flow rate, Contact time: 420 s, Flow rate: 10 μl / min. Temperature: 25°C. Coupling reagents: 100 μl each of EDC and NHS (from the amine coupling kit). Block with 140 μl of ethanolamine (from the amine coupling kit). Running buffer: 1× HBS-EP. The coupling amount of STAT3 is approximately 30,000 RU. As shown in the results, the STAT3 coupling amount on Chip CM7 is higher than that on Chip CM5.

[0194] 2.8 SPR testing of different batches of Angong Niuhuang Wan

[0195] SPR screening of STAT3 was performed based on 3 batches of Angong Niuhuang Wan and 5 batches of homemade samples.

[0196] 2.8.1 Preparation of test samples

[0197] Preparation of Angong Niuhuang Wan solution: Take 0.1g of Angong Niuhuang Wan pills, place them in a 25ml conical flask, add 10ml of 80% methanol, ultrasonically extract for 30min (50kHz, 350W), make up the weight loss with 80% methanol, centrifuge at 14000r for 10min, accurately aspirate 2ml of supernatant, centrifuge and concentrate at 4℃ to dryness, add 200μl DMSO to dissolve it, filter through 0.22μm pore membrane, accurately aspirate 10μl, add 190μl PBS solution, and mix well.

[0198] Preparation of homemade intermediate solution: take 0.1g bezoar, 0.2g buffalo horn concentrated powder, 0.025g musk, 0.05g pearl, 0.1g cinnabar, 0.1g realgar, 0.1g coptis powder, 0.1g scutellaria powder, 0.1g gardenia powder, 0.1g turmeric powder, and 0.025g borneol, place them in a 250ml conical flask, add 100ml of 80% methanol, ultrasonically extract for 30min (50kHz, 350W), make up the weight loss with 80% methanol, centrifuge at 14000r for 10min, accurately aspirate 2ml of supernatant, centrifuge and concentrate at 4℃ to dryness, add 200μl DMSO to dissolve, filter through 0.22μm pore membrane, accurately aspirate 10μl, add 190μl PBS solution, and mix well.

[0199] Preparation of the positive agent berberine: Accurately weigh an appropriate amount of berberine and prepare a 10 mM stock solution with DMSO. Add 5 μl of the control stock solution to 95 μl of 1x PBS and 900 μl of 5% DMSO in PBS, and mix thoroughly to a 50 μM solution in 5% DMSO in PBS. Chlorogenic acid, a negative agent, is prepared in the same manner.

[0200] 2.8.2 SPR Detection of STAT3 Protein in Three Batches of Angong Niuhuang Wan and Five Batches of Homemade Intermediates

[0201] 100 μl aliquots of each test solution from three batches of Angong Niuhuang Wan samples, five batches of a homemade intermediate, the positive drug berberine, and the negative drug chlorogenic acid were precisely pipetted into a 96-well Biacore assay plate. The assay was run with a contact time of 60 seconds, a flow rate of 30 μl / min, a dissociation time of 60 seconds, and a temperature of 25°C. The running buffer was PBS containing 5% DMSO. The ratios of the RU values ​​of the three batches of Angong Niuhuang Wan and the five batches of the homemade intermediate to the RU value of berberine are shown in Table 4. The results show that the ratios were at least greater than 1.

[0202] Table 4 Ratios of RU values ​​of three batches of Angong Niuhuang Wan and five batches of homemade intermediates to the RU value of positive berberine

[0203]

[0204]

[0205] 3. Discussion

[0206] Finally, based on methodological considerations, the CM7 chip was selected because it has a higher protein coupling capacity. The test sample was extracted with 80% methanol and dissolved in DMSO at a ratio of 1:10 (g / ml). Berberine was selected as the control sample at a concentration of 50 μM.

[0207] In summary, the quality evaluation method of Angong Niuhuang Pills based on the biological effect of STAT3 protein in the present invention is simple, easy to operate, and highly accurate. It can reflect the effectiveness of Angong Niuhuang Pills in clinical treatment, further improve the quality control system of Angong Niuhuang Pills, and has practical application and promotion value.

Claims

1. A method for evaluating the quality of Angong Niuhuang Wan based on the biological effect of STAT3 protein, characterized by: It uses SPR detection, which includes the following steps: 1) Construction of protein chip: Dissolve STAT3 protein in water, then dilute with sodium acetate buffer to a 10-40 μg / ml protein solution. The protein solution is coupled to the sensor chip. 2) Preparation of test solution: Extract Angong Niuhuang Wan with 80% methanol solution, centrifuge the extract, dry the supernatant, reconstitute with DMSO, filter, and mix the filtrate with PBS solution. 3) Preparation of reference solution: Dissolve berberine in DMSO, add PBS solution and DMSO-containing PBS solution to the dissolved solution and mix well. 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); SPR assay conditions: run time: 60 s; flow rate: 30 μl / min; dissociation time: 60 s; temperature: 25°C; running buffer: 5% DMSO in PBS; The ratio of the RU value of Angong Niuhuang Wan detected by SPR to the RU value of berberine is not less than 1.

2. The method according to claim 1, wherein: Step 1) The protein solution is 20 μg / ml.

3. The method according to claim 1, wherein: Step 1) The coupling is amino coupling; the parameters of the amino coupling are: sensor chip model: CM7 or CM5, contact time: 420 s, flow rate: 10 μl / min, temperature: 25°C, coupling reagents: EDC and NHS, blocking reagent: ethanolamine, running buffer: 1×HBS-EP.

4. The method according to claim 3, wherein: The sensor chip model is CM7; the volume ratio of the protein solution, EDC, NHS, and ethanolamine is 100:200:140; and the volume ratio of EDC to NHS is 1:

1.

5. The method according to claim 1, wherein: Step 2) The mass volume ratio of the Angong Niuhuang Pills to the 80% methanol solution is 0.1 g: 5-25 ml; the extraction is ultrasonic extraction, the time is 20-60 min, the power is 20-80 kHz, 100-500 W; the centrifugation speed is 5000-25000 r, the time is 5-25 min; the amount of DMSO added is 15-25 times that of the supernatant.

6. The method according to claim 1, wherein: Step 3) The concentration of berberine 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.

7. The method according to claim 1, wherein: The DMSO concentration in the PBS solution containing DMSO is 5%.

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