Method for analyzing multiple components in methylstilbene liver fiber particles

Abstract

The invention belongs to the technical field of chemical component analysis, and provides a method for analyzing multiple components in methylstilbene liver fiber granules, which comprises the following steps: (1) preparing a test solution: extracting methylstilbene liver fiber granules with a methanol solution to obtain the test solution; (2) preparing reference substance solutions: taking 20 reference substances such as betaine, adenine, gallic acid and the like, and respectively adding methanol to prepare the reference substance solutions; (3) detecting and analyzing the test sample solution and the reference substance solution by adopting a triple quadrupole flight time high-resolution mass spectrum; and (4) collecting data and analyzing, so as to analyze and identify the components of the methylstilbene liver fiber particles. The chemical components of the methylstilbene liver fiber granules are analyzed and identified by adopting a UPLC-Q-TOF-MS method, and a basis is provided for further researching the pharmacodynamic material basis of the methylstilbene liver fiber granules.

Description

Technical Field

[0001] This invention relates to the field of chemical component analysis technology of traditional Chinese medicine, specifically to an analytical method for multiple components in Jiaqi Liver Fibrosis Granules. Background Technology

[0002] Jiuzhitang Pharmaceutical Co., Ltd.'s proprietary traditional Chinese medicine product, Jiaqi Liver Fibrosis Granules [Standard No.: WS3-737(Z-203)-2006(Z)-2007], is composed of 11 Chinese medicinal herbs including Astragalus membranaceus, Stephania tetrandra, and Poria cocos. It has the effects of soothing the liver, promoting blood circulation, strengthening the spleen, and removing dampness. Clinically, it is used to treat hepatitis B liver fibrosis with abnormal serum indicators such as hyaluronic acid, type IV collagen, and laminin, belonging to the category of liver stagnation and blood stasis combined with spleen deficiency and dampness retention, manifested as hypochondriac pain, hepatosplenomegaly, abdominal distension, fatigue, poor appetite, loose stools, dark purple tongue or with ecchymosis, and greasy tongue coating. Currently, pharmacological and pharmacodynamic research on Jiaqi Liver Fibrosis Granules is relatively weak. Therefore, further exploration of the main chemical components of Jiaqi Liver Fibrosis Granules is very beneficial for its material basis research and pharmacological and pharmacodynamic studies. Summary of the Invention

[0003] The purpose of this invention is to address the current weakness in research on the material basis, pharmacology, and efficacy of Jiaqi Ganxian Granules by providing an analytical method for multiple components in Jiaqi Ganxian Granules. This invention uses UPLC-Q-TOF-MS to analyze and identify the chemical components in Jiaqi Ganxian Granules, providing a basis for further research on the material basis of the efficacy of Jiaqi Ganxian Granules.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] An analytical method for multiple components in Astragalus membranaceus liver-fiber granules includes the following steps:

[0006] (1) Preparation of test solution: Take Astragalus membranaceus liver fibrinolysis granules and extract them with methanol solution to obtain test solution;

[0007] (2) Preparation of reference solutions: Take 20 reference standards, including betaine, adenine, gallic acid, amygdalin, paeoniflorin, magnoflorine, paeoniflorin, galloylpaeoniflorin, fangchinoline, corydaline, tetrandrine, gentianin, corydaline A, verbascoflavonoids, benzoylpaeoniflorin, astragaloside A, gentianin, turmeric dione, magnolol, and honokiol, and prepare standard solutions by adding methanol to each. Take an appropriate amount from each of the 20 standard solutions and mix them evenly to obtain the reference solution; (3) Detect and analyze the test solution and reference solution by high-resolution mass spectrometry.

[0008] (4) Analyze the data collected by mass spectrometry and identify the components of Jiaqi liver fiber granules.

[0009] The chromatographic conditions were as follows: a C18 column, preferably an Agilent ZORBAX Eclipse Plus C18 (3.0 × 100 mm, 1.8 μm); and a mobile phase consisting of an organic phase acetonitrile (A) and an aqueous phase (B), wherein the aqueous phase B contained 0.05 mmol / L. -1 Formic acid, set the gradient elution program as follows: 0–10 min, 5–15% A; 10–20 min, 15%–35% A; 20–30 min, 35%–55% A; 30–38 min, 55%–85% A; flow rate: 0.4 mL / min, injection volume: 2 μL.

[0010] The mass spectrometry conditions were as follows: positive and negative ion analysis mode, electrospray ionization (ESI) as the ionization method, primary mass spectrometry scanning detection range: m / z 100~1700, nitrogen as the solvent-removing drying gas, temperature 325℃, flow rate 6.8L / min; sheath gas temperature 350℃; capillary voltage 4.0kV, fragment voltage 150V.

[0011] Preferably, the mass number is calibrated using Agilent standard tuning solution ESI-L Low Concentration Tuning Mix (G1969-85000) before mass spectrometry injection analysis.

[0012] Preferably, the ratio of Astragalus membranaceus liver fiber granules to methanol solution is 3g:30ml.

[0013] This invention is the first to perform comprehensive chemical composition analysis of Jiaqi Ganxian Granules using UPLC-Q-TOF-MS. By optimizing the extraction, chromatographic, and mass spectrometric conditions for the active ingredients, a highly efficient method for separating multiple active ingredients was established under both positive and negative ion modes. Through activity matching with a database, the chemically active components of the sample were qualitatively analyzed, identifying 116 active ingredients and assigning their drug sources. This invention, using UPLC-Q-TOF-MS technology, preliminarily clarifies the main chemical component groups of Jiaqi Ganxian Granules, laying the foundation for subsequent research on the pharmacodynamic material basis, quality control, and promotion of clinical applications. Attached Figure Description

[0014] Figure 1 Total ion current spectrum (positive ion mode) of Astragalus membranaceus liver fibrinolysis granules

[0015] Figure 2 Total ion current spectrum (negative ion mode) of Astragalus membranaceus liver fibrinolysis granules

[0016] Figure 3Total ion chromatogram of traditional Chinese medicine reference standards (C-positive ion mode; D-negative ion mode;) Detailed Implementation

[0017] An analytical method for multiple components in Astragalus membranaceus liver fiber granules. To more clearly illustrate the present invention, the following specific embodiments are provided for further explanation.

[0018] 1. Instruments and Materials

[0019] Agilent 1290UPLC-6540Q-TOF chromatography-mass spectrometry system (Agilent Technologies, USA, equipped with a MassHunter mass spectrometry workstation and Mass Analysis mass spectrometry data processing system, Agilent ZORBAX EclipsePlus C18 (3.0×100mm, 1.8μm) column; ultrasonic instrument (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), S-23 high-speed benchtop centrifuge (Hunan Xiangyi Instrument Co., Ltd.); AUW120D electronic analytical balance (SHIMADZU, Japan); chromatographic methanol and mass spectrometric acetonitrile (both purchased from Merck Millipore, Germany); distilled water (Shenzhen Watson Distilled Water Co., Ltd.); ammonium formate (mass spectrometry grade, Fisher Scientific, USA); reagents: Astragalus and Astragalus Liver Fibrosis Granules (Jiuzhitang Pharmaceutical Co., Ltd., batch numbers: 20230905, 20240306, 20240307), information on traditional Chinese medicine reference standards is shown in Table 1;

[0020] Table 1. Reference Standard Information

[0021] Serial Number name Manufacturer batch number 1 betaine China National Institutes for Food and Drug Control 110894-201604 2 adenine Baoji Chenguang Biotechnology Co., Ltd. HR2792W3 3 gallic acid China National Institutes for Food and Drug Control 111672-200401 4 amygdalin China National Institutes for Food and Drug Control 110820-201808 5 Paeoniflorin Beijing Solarbio Technology Co., Ltd. 20220419 6 Magnolia alkaloids Chengdu Pufeed Biotechnology Co., Ltd. 21081902 7 paeoniflorin National Institutes for Food and Drug Control 110736-202246 8 Galloylpaeoniflorin Shanghai Shidander Standard Technical Service Co., Ltd. ST23740110 9 Tetrandrine National Institutes for Food and Drug Control 110793-202108 10 Corydalis B National Institutes for Food and Drug Control 11076-202020 11 Stephania tetrandra Chengdu Pufeed Biotechnology Co., Ltd. 20120404 12 Mangbinghua glycoside Chengdu Pufeed Biotechnology Co., Ltd. 21020145 13 Corydalis A Shanghai Shidander Standard Technical Service Co., Ltd. ST05600120 14 Versicolor isoflavones Chengdu Pufeed Biotechnology Co., Ltd. 21032501 15 Benzoylpaeoniflorin Beijing Yashier Technology Co., Ltd. 190719-053 16 Astragaloside A National Institutes for Food and Drug Control 110781-202117 17 Ammonium truncatum Shanghai Tongtian Biotechnology Co., Ltd. 21071531 18 Curcumin Chengdu Pufeed Biotechnology Co., Ltd. 21092901 19 honokiol National Institutes for Food and Drug Control 11730-201915 20 magnolol National Institutes for Food and Drug Control 11729-202015

[0022] 2 Experimental Methods

[0023] 2.1 Collection of chemical components of Astragalus Liver-Fibrin Granules and construction of a database

[0024] Based on existing data on Chinese herbal ingredients in our laboratory, the database of the Encyclopedia of Traditional Chinese Medicine, and literature review, we collected the chemical components of 11 Chinese herbs in Jiaqi Liver Fibrosis Granules: Astragalus membranaceus, Stephania tetrandra, Poria cocos, Magnolia officinalis, Corydalis yanhusuo, Paeonia lactiflora, Achyranthes bidentata, Prunus persica, Curcuma zedoaria, Trionyx sinensis, and Eupolyphaga sinensis. The main information includes compound name, molecular formula, molecular weight, and compound structure.

[0025] 2.2 LC-MS Analysis of Astragalus-Astragalus Liver Fibrosis Granules

[0026] 2.2.1 Chromatographic and Mass Spectrometry Conditions

[0027] Chromatographic conditions: Agilent ZORBAX Eclipse Plus C18 column (3.0×100mm, 1.8μm).

[0028] The mobile phase consists of an organic phase acetonitrile (A) and an aqueous phase (B), wherein the aqueous phase B contains 0.05 mmol·L⁻¹. -1 Formic acid (mass spectrometry grade), with a gradient elution program set as follows: 0–10 min, 5–15% A; 10–20 min, 15%–35% A; 20–30 min, 35%–55% A; 30–38 min, 55%–85% A; flow rate: 0.4 mL / min, injection volume: 2 μL.

[0029] Mass spectrometry detection conditions: Positive and negative ion analysis modes were used, with electrospray ionization (ESI) as the ionization method. Before sample injection and analysis, Agilent standard tuning solution ESI-L Low Concentration TuningMix (G1969-85000) was used for accurate mass number calibration. The primary mass spectrometry scanning detection range was m / z 100–1700, using nitrogen as the solvent-removing drying gas at a temperature of 325℃ and a flow rate of 6.8 L / min; the sheath gas temperature was 350℃; the capillary voltage was 4.0 kV, and the fragment voltage was 150 V.

[0030] 2.2.2 Solution Preparation

[0031] 2.2.2.1 Preparation of Astragalus-Methionine Liver Fibrosis Granules Test Solution

[0032] Accurately weigh 3.0 g of Astragalus membranaceus liver fibrillation granules and place them in a 50 ml stoppered conical flask. Add 30 ml of methanol solution to each flask, weigh, and then extract by sonication for 30 min. Allow to stand at room temperature, make up the weight loss, and let stand. Take the supernatant, centrifuge at 8000 r / min for 5 min, and filter through a 0.22 μm microporous membrane to obtain the Astragalus membranaceus liver fibrillation granule sample solution for analysis.

[0033] 2.2.2.2 Preparation of the reference solution

[0034] Accurately weigh 10 mg each of the following 20 reference standards: betaine, adenine, gallic acid, amygdalin, paeoniflorin, magnoflorine, paeoniflorin, galloylpaeoniflorin, tebufenozide, corydaline, gentianin, corydaline A, verbascoflavonoids, benzoylpaeoniflorin, astragaloside A, gentianin, turmeric dione, magnolol, and honokiol. Place each standard in a 25 mL volumetric flask, add 20 mL of methanol, sonicate to dissolve, shake well, and then add methanol to the mark to obtain the standard stock solution. Take an appropriate amount of the reference standard from each of the 20 standard solutions and mix them evenly to obtain mixed reference solution 1 (used for qualitative analysis and identification of the chemical components of Jiaqi liver fibrinolysis granules).

[0035] 2.2.3 LC-MS Analysis and Data Acquisition

[0036] A UPLC-Q-TOF-MS-based liquid chromatography-mass spectrometry method was established to detect and acquire data of chemical components in Astragalus membranaceus liver fibrillation granules, employing full scan-dynamic exclusion and data-dependent scanning. Molecular feature extraction (MFE) was used in the Agilent Masshunter Qualitative Analysis workstation. Compounds with an absolute peak height >5000 and a relative peak height >1.5% were extracted and analyzed to obtain information such as chromatographic retention time, quasi-molecular ion peaks, fragment ions, and molecular formula.

[0037] 2.3 Data analysis and component identification of the chemical composition of Astragalus membranaceus liver fiber granules

[0038] Qualitative analysis was performed based on the primary and secondary mass spectrometry data and mass spectrometry fragment ion information of the compound provided by mass spectrometry scanning. The molecular formula of the compound was predicted by accurate molecular weight, and identification was performed by combining relevant mass spectrometry fragmentation patterns, literature data, multi-stage mass spectrometry fragment information, and comparison with traditional Chinese medicine reference standards.

[0039] 3 Experimental Results

[0040] 3.1 Construction of a database of chemical components of Astragalus membranaceus liver fibrosis granules

[0041] Chemical components of 11 Chinese herbs in Jiaqi Ganxian Granules were searched using the Taiwan Chinese Medicine Database (http: / / tcm,cmu,edu.tw / ), the Chinese Materia Medica Systems Pharmacology Database and Analysis Platform of Northwest A&F University (http: / / lsp.nwu.edu.cn / tcmsp.php), the Chinese Medicine Bioinformatics Database of the School of Life Sciences of East China Normal University (http: / / www.megabionet.org / tcmid / ), and electronic databases such as PubMed, SciFinder, and CNKI. A total of 1572 chemical components were collected, including 356 from Astragalus membranaceus, 125 from Stephania tetrandra, 179 from Poria cocos, 174 from Magnolia officinalis, 102 from Corydalis yanhusuo, 191 from Paeonia lactiflora, 87 from Achyranthes bidentata, 141 from Prunus persica, 140 from Curcuma zedoaria, 36 from Trionyx sinensis, and 64 from Eupolyphaga sinensis. The compound information included chemical component name, molecular formula, and molecular weight.

[0042] 3.2 Chemical composition analysis of Astragalus membranaceus liver fibrosis granules

[0043] A UPLC-Q-TOF-MS-based liquid chromatography-mass spectrometry method was established to detect and collect data on the chemical components in the test and reference solutions of Jiaqi Liver Fibrosis Granules using a full-scan, dynamic exclusion, and data-dependent scanning approach. Molecular Feature Extraction (MFE) was employed in the Agilent Masshunter Qualitative Analysis workstation. Compounds with an absolute peak height >5000 and a relative peak height >1.0% were extracted and analyzed to obtain chromatographic retention time, quasi-molecular ion peaks, fragment ions, and molecular formula. Qualitative analysis was performed based on the primary and secondary mass spectrometry data and fragment ion information provided by the mass spectrometry scans. The molecular formula was predicted by accurate molecular weight, and rapid identification was achieved by combining relevant mass spectrometry fragmentation patterns, literature data, multi-stage mass spectrometry fragment information, and comparison with traditional Chinese medicine reference standards.

[0044] A total of 116 compounds were rapidly identified from Jiaqi Liver Fibrosis Granules, mainly flavonoids, alkaloids, steroids, amino acids, fatty acids, and phenolic acids. The sources of these compounds were attributed to various sources, including 24 from Astragalus membranaceus, 5 from Stephania tetrandra, 9 from Poria cocos, 18 from Magnolia officinalis, 14 from Corydalis yanhusuo, 14 from Paeonia lactiflora, 9 from Achyranthes bidentata, 14 from Prunus persica, 4 from Curcuma zedoaria, 15 from Trionyx sinensis, and 12 from Eupolyphaga sinensis. UPLC-Q-TOF-MS analysis was performed on these compounds. n The total ion chromatograms under positive and negative ion modes are shown in 1-3. The retention time, molecular formula, compound name, high-resolution mass spectrometry parent ion and difference, fragment ion information, and medicinal material source of the chemical components of Jiaqi liver fibrin granules are shown in Table 2.

[0045]

[0046]

[0047]

[0048]

Claims

1. A method for analyzing multiple components in Astragalus membranaceus liver-fiber granules, characterized in that, Includes the following steps: (1) Preparation of test solution: Take Astragalus membranaceus liver fibrinolysis granules and extract them with methanol solution to obtain test solution; (2) Preparation of reference solutions: Take 20 reference standards, namely betaine, adenine, gallic acid, amygdalin, paeoniflorin, magnoflorine, paeoniflorin, galloylpaeoniflorin, fentanyl, tetrandrine, gentianin, gentianin, verbascoside, benzoylpaeoniflorin, astragaloside A, gentianin, turmeric dione, magnolol, and honokiol, and prepare standard solutions by adding methanol to each of the 20 standard solutions. Take an appropriate amount from each of the 20 standard solutions and mix them evenly to obtain the reference solution. (3) The test solution and the reference solution were analyzed by high-resolution mass spectrometry. (4) Analyze the data collected by mass spectrometry and identify the components of Jiaqi liver fiber granules.

2. The analytical method according to claim 1, characterized in that, The chromatographic conditions were as follows: a C18 column was used, and the mobile phase consisted of an organic phase of acetonitrile (A) and an aqueous phase (B), wherein the aqueous phase B contained 0.05 mmol / L. -1 Formic acid, gradient elution program: 0–10 min, 5–15% A; 10–20 min, 15%–35% A; 20–30 min, 35%–55% A; 30–38 min, 55%–85% A; flow rate: 0.4 mL / min, injection volume: 2 μL.

3. The analytical method according to claim 1, characterized in that, The mass spectrometry conditions were as follows: positive and negative ion analysis mode was used, the ionization method was electrospray ionization (ESI), the detection range of the first-order mass spectrometry scan was 100 to 1700 m / z, nitrogen was used as the solvent removal and drying gas, the temperature was 325℃, the flow rate was 6.8 L / min, the sheath gas temperature was 350℃, the capillary voltage was 4.0 kV, and the fragment voltage was 150 V.

4. The analytical method according to claim 3, characterized in that, Before mass spectrometry analysis, Agilent standard tuning solution ESI-LLow Concentration Tuning Mix (G1969-85000) was used to perform accurate mass number calibration.

5. The analytical method according to claim 1, characterized in that, In the preparation of the test sample, the ratio of Astragalus membranaceus liver fibrinolytic granules to methanol solution was 3g:30ml.

Images