Method for detecting material basis of total components of Xiaoming tablet extract

Abstract

The invention discloses a method for detecting the material basis of all components of an extract of Xiaoyingming tablets. The preparation method comprises the following steps: A, preparing a Tablet extract, and preparing the Tablet extract into a test solution; b, preparing a reference medicinal material solution, wherein reference medicinal materials comprise semen cuscutae, radix rehmanniae praeparata, fructus lycii, radix codonopsis and radix astragali seu hedysari; c, preparing a reference solution; and D, carrying out high performance liquid chromatography-mass spectrometry analysis on the test solution, the reference medicinal material solution and the reference substance solution to obtain total-component substances of the Xiaoming tablet extract. According to the method, corresponding components of the twenty-two medicinal materials in the prescription of the Xiaoyanming tablet can be detected, the whole composition prescription can be covered, the component detection is relatively comprehensive, and the coverage is wide.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and particularly relates to a method for detecting the material basis of all components of Zhanyanming tablets extract. Background Art

[0002] The prescription of Zhanyanming has 40 years of experience in medicine making. Its tablet prescription comes from Professor Li Zaoyun, a national ophthalmologist and famous Chinese medicine expert. It is developed based on the traditional prescriptions Yiqi Congming Decoction and Zhujing Pills, combined with its own medication experience, and recorded in the "Handbook of Practical Basic Chinese Medicine Preparations". It is now included in the 2020 edition of the Chinese Pharmacopoeia Volume 1.

[0003] Zhanyanming Pian is a compound Chinese patent medicine composed of 22 Chinese herbal medicines, mainly including Cuscuta chinensis, Rehmannia glutinosa, Codonopsis pilosula, Lycium barbarum, Astragalus membranaceus, Buddleja scutellariae, Chrysanthemum, Ligusticum chuanxiong, Pueraria lobata and other medicinal materials. It has the effects of nourishing the liver and kidneys, removing cataracts and improving eyesight. Among them, Cuscuta chinensis and Rehmannia glutinosa nourish the liver and kidneys, improve essence and improve eyesight; Codonopsis pilosula and Lycium barbarum assist the main medicine to enhance the effect of nourishing the liver and kidneys, improving essence and improving eyesight; Chrysanthemum and Buddleja scutellariae clear the liver, improve eyesight, remove cataracts and remove cataracts. Summary of the Invention

[0004] The present invention aims to establish a detection method that can monitor the chemical composition of all substances in the Zhanyanming tablets extract, thereby laying a material foundation for the further development and utilization of the Zhanyanming tablets.

[0005] The present invention aims to comprehensively reveal the chemical basis of Zhanyanming tablets. By establishing a preparation method for drug sample test products, liquid chromatography and mass spectrometry conditions, the UFLC-Triple Q-TOF-MS / MS technology is used to perform online separation and identification of all chemical components of the Zhanyanming tablet extract. The structures of the chemical components are identified through methods such as precise molecular weight and isotope matching of primary mass spectrometry, comparison with reference substances through secondary mass spectrometry fragmentation patterns, library retrieval (NCBIPubChem Database, ChEMBL, Chem-spider, etc.), and comparison with relevant literature. High-resolution TOF-MS primary data is used for quantitative analysis of chemical components, completing the characterization of the full chemical components of Zhanyanming tablets and establishing its chemomics database.

[0006] The method for detecting the material basis of all components of the Zhanyanming tablet extract of the present invention comprises the following steps:

[0007] A. Prepare the extract of Zhanyanming tablets and make the test solution of the extract of Zhanyanming tablets;

[0008] B. Prepare a control medicinal material solution; the control medicinal materials are Cuscuta chinensis, Rehmannia glutinosa, Lycium barbarum, Codonopsis pilosula, and Astragalus membranaceus;

[0009] C. Prepare reference solution;

[0010] D. Perform high performance liquid chromatography-mass spectrometry analysis on the test sample solution, control medicinal material solution and reference substance solution to obtain the complete components of the Zhanyanming tablets extract.

[0011] Preferably, the HPLC conditions are:

[0012] A Thermo (250*4.6mm*5μm) column was used, 0.1% formic acid aqueous solution was used as aqueous phase A, and mass spectrometry grade acetonitrile was used as organic phase B. The gradient elution was as follows:

[0013]

[0014]

[0015] The flow rate was 1.0 mL / min and the column temperature was 35°C.

[0016] Preferably, the mass spectrometry conditions are as follows: electrospray ion source, positive and negative ion mode detection, in positive ion mode, the spray voltage is set to 5500V; in negative ion mode, it is -4500V, the spray pressure is set to 55psi, the auxiliary heating pressure is 50psi, the ion source temperature is maintained at 550°C, the collision voltage is 30V, the declustering voltage is 80V, the air curtain pressure is 35psi, the collider pressure is 10psi, the scanning range covers m / z 50-1500, nitrogen is used as the spray gas and auxiliary gas, and The software collects data in Information Dependent Acquisition (IDA) mode.

[0017] Preferably, the test solution is prepared as follows:

[0018] Weigh 0.5 g of the extract of Zhanyanming tablets, dissolve it in a 25 mL volumetric flask, make up to volume with 75% methanol solution and weigh it, ultrasonicate for 30 min, add to the original weight, and filter through a 0.45 μm microporous filter membrane to obtain the product.

[0019] Preferably, the preparation method of the control medicinal material solution is as follows:

[0020] Weigh 6.1 g of Cuscuta seeds, 6.1 g of Rehmannia roots, 4.8 g of Lycium barbarum, 4.8 g of Codonopsis pilosula, and 4.8 g of Astragalus membranaceus, soak them in 100 mL of boiling water for 20 minutes each, and decoct them for 1 hour. Decoction them twice in total, combine the filtrates, concentrate them to 5 mL, and filter them through a 0.45 μm microporous membrane.

[0021] Preferably, the preparation method of the reference solution is as follows:

[0022] Weigh 5 mg of reference substance into a volumetric flask, add 25 mL of methanol to each flask and dilute to volume. Dissolve until clear and transparent to obtain a 200 μg / mL reference substance mother solution. Take 5 μL of each reference substance mother solution and mix them. Add methanol to 200 μL and mix evenly.

[0023] Preferably, the full component analysis is shown in Table 3:

[0024] The method of the present invention has the following advantages:

[0025] Comprehensive coverage: The corresponding ingredients of the twenty-two medicinal materials in the prescription of Zhanyanming Tablets have been detected, which can cover all the components of the prescription. The detection of its ingredients is relatively comprehensive and has a wide coverage.

[0026] Key points: Among the detected components, more than 60% can be attributed to the main and auxiliary medicinal materials, indicating that this method has good coverage and specificity for core characteristic medicinal materials, is highly practical, and is scientific and reasonable.

[0027] Simple and fast: The entire detection time is 50 minutes, and readily available organic solvents are used as the mobile phase. The detection method is simple, safe, fast and efficient.

[0028] Good separation: Substances are detected throughout the entire time period, and the components are well separated, so the test results are relatively scientific and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the total ion chromatogram of the blank solvent.

[0030] Figure 2 This is the total ion current diagram of the Zhanyanming tablet extract.

[0031] Figure 3 It is the classification of the mother nucleus of the compound of the Zhanyanming tablet extract.

[0032] Figure 4 It is the medicinal material of the compound of Zhanyanming tablet extract;

[0033] Figure 5 It is a classification diagram of the parent nucleus of compound components;

[0034] Figure 6 It is a diagram of the classification of compound components and medicinal materials. DETAILED DESCRIPTION

[0035] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0036] Example 1:

[0037] 1. Experimental Methods

[0038] 1. Test solution preparation:

[0039] The extract of Zhanyanming tablets (Guangzhou Baiyunshan Zhongyi Pharmaceutical, CA1007) is available in the preparation method of Zhanyanming tablets in Part 1 of the 2020 edition of the Chinese Pharmacopoeia. The crude drug content is 2.48 g / g.

[0040] Weigh 0.5 g of the extract of Zhanyanming tablets and dissolve it in a 25 mL volumetric flask. Dose the solution with 75% methanol solution, then weigh the solution. After sonication for 30 minutes, bring the volume to the original weight and filter through a 0.45 μm microporous filter. 10 μL of the filtrate was injected into ultra-fast liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry to determine the chemical components of the extract.

[0041] 2. Preparation of control medicinal material solution:

[0042] Weigh 6.1 g of Cuscuta seeds, 6.1 g of Rehmannia root, 4.8 g of Lycium barbarum, 4.8 g of Codonopsis pilosula, and 4.8 g of Astragalus membranaceus. Soak each in 100 mL of boiling water for 20 minutes each, then decoct for 1 hour. Repeat this decoction twice. Combine the filtrates, concentrate to 5 mL, and filter through a 0.45 μm microporous filter. 10 μL of the filtrate was injected into ultra-fast liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry for chemical component analysis.

[0043] 3. Preparation of reference solution:

[0044] Accurately weigh 5 mg of each reference substance into a volumetric flask, add 25 mL of methanol and dilute to volume, dissolve until clear and transparent to obtain a 200 μg / mL reference substance mother solution, take 5 μL of each reference substance mother solution and mix, add methanol to 200 μL and mix evenly, take 10 μL for injection.

[0045] 4. UFLC-Q-TOF-MS / MS chromatographic conditions:

[0046] Column temperature:

[0047] A Thermo (250*4.6mm*5μm) column was used, with 0.1% formic acid aqueous solution as aqueous phase A and mass spectrometry-grade acetonitrile as organic phase B. Gradient elution (elution conditions are shown in Table 1) with a flow rate of 1.0mL / min and column temperatures of 25℃, 35℃, and 40℃ was used to investigate the effect of injection temperature on the experimental results.

[0048] Table 1 Liquid phase elution gradient

[0049]

[0050] As the temperature increases, the elution time of each component becomes shorter and the elution rate increases. At 35℃ and 40℃, the peak time of each component is similar, and the peak shape of each component is better at 35℃. Therefore, 35℃ is selected as the column temperature condition. The chromatographic results are shown in Figure 2. Figure 1 .

[0051] Liquid phase elution gradient:

[0052] When investigating the column temperature, it was found that there were still peaks of substances in the column equilibrium period (40-45 minutes). Therefore, it was considered to extend the elution time and increase the elution ratio of the organic phase from 75% to 90% to make the substances in the column equilibrium period elute earlier. The chromatographic results are shown in Figure 2. Figure 2 .

[0053] Therefore, the optimal elution conditions are: using a Thermo (250*4.6mm*5μm) as the chromatographic column, 0.1% formic acid aqueous solution as the aqueous phase A, mass spectrometry-grade acetonitrile as the organic phase B, gradient elution (elution conditions are shown in Table 2), a flow rate of 1.0mL / min, and a column temperature of 35℃.

[0054] Table 2 Liquid phase elution gradient

[0055]

[0056]

[0057] 5. UFLC-Q-TOF-MS / MS mass spectrometry parameters:

[0058] Electrospray ionization (ESI) was used for detection in positive and negative ion modes. In positive ion mode, the spray voltage was set to 5500 V; in negative ion mode, it was -4500 V. The spray pressure was set to 55 psi, and the auxiliary heating pressure was 50 psi. The ion source temperature was maintained at 550 °C, the collision voltage was 30 V, the declustering voltage was 80 V, the curtain pressure was 35 psi, and the collider pressure was 10 psi. The scan range covered m / z 50-1500. Nitrogen was used as the spray gas and auxiliary gas, and The software collects data in Information Dependent Acquisition (IDA) mode.

[0059] 2. Experimental Results

[0060] In this study, ultra-performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (UPLC-Triple-TOF-MS / MS) was used to analyze the full composition of the Zhanyanming tablet extract and its corresponding medicinal materials: Rehmannia root, Cuscuta seed, Lycium barbarum fruit, Codonopsis pilosula, and Astragalus membranaceus. The specific composition information is shown in Table 3.

[0061] The mass spectrometry blank spectrum and total ion current spectrum are as follows Figure 3 and Figure 4 As shown:

[0062] Through reference substance comparison, accurate molecular weight comparison, pyrolysis fragment analysis and spectral library search matching, 215 compounds in Zhanyanming tablets were confirmed and identified, and divided into 13 major categories, including 43 flavonoids, 32 terpenes, 24 phenylpropanoids, 23 alkaloids, 16 organic acids, 16 phenylethanol glycosides, 12 sugars and glycosides, 11 triterpenoid saponins, 11 fatty acids and fatty acid esters, 8 amino acids, 7 quinones, 6 phthalides and 6 other compounds. The compound component parent nucleus classification diagram is shown in Figure 5 .

[0063] By comparing the monarch and ministerial herbs and analyzing the references, 46 compounds were identified as belonging to Cuscuta chinensis, 41 compounds as belonging to Rehmannia glutinosa, and 22 compounds as belonging to Fructus Lycii, 21 compounds as belonging to Codonopsis pilosula, 13 compounds as belonging to Astragalus membranaceus, 17 compounds as belonging to Semen Plantaginis, 15 compounds as belonging to Rhizoma Chuanxiong, 14 compounds as belonging to Paeonia lactiflora, 12 compounds as belonging to Cistanche deserticola, 12 compounds as belonging to Chrysanthemum Flos, 11 compounds as belonging to Buddlejac, 10 compounds as belonging to Semen Cassiae, 10 compounds as belonging to Fructus Viticis, 9 compounds as belonging to Phellodendron chinense, 8 compounds as belonging to Fructus Corni, 7 compounds as belonging to Fructus Amaranthus, 4 compounds as belonging to Radix Puerariae, 4 compounds as belonging to Acorus gramineus, 4 compounds as belonging to Rhizoma Rugosae, 4 compounds as belonging to Cimicifuga heracleifolia, 3 compounds as belonging to Polygonatum sibiricum, and 10 compounds as belonging to Glycyrrhiza uralensis.

[0064] Among them, more than 65% of the chemical components can be classified as monarch and ministerial drugs, with monarch drugs accounting for 40.47% and ministerial drugs accounting for 26.05%. Figure 6 .

[0065] summary:

[0066] This study identified 215 chemical components in the extract of Zhanyanming tablets, nearly 60% of which belong to flavonoids, terpenes, phenylpropanoids, and alkaloids. This suggests that subsequent quality improvement efforts should focus on monitoring and optimizing these three characteristic components. Medicinal material attribution analysis revealed that over 60% of the identified components belong to the sovereign and auxiliary medicinal materials, demonstrating both good coverage and specificity for the core characteristic medicinal materials, and a highly practical and scientifically sound approach.

[0067] Table 3 Analysis of the full components of the extract of Zhanyanming tablets

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

Claims

1. A method for detecting the material basis of all components of Zhanyanming tablets extract, characterized in that: The following steps are involved: A. Prepare the extract of Zhanyanming tablets and make the test solution of the extract of Zhanyanming tablets; B. preparing a control medicinal material solution, wherein the control medicinal materials are Cuscuta chinensis, Rehmannia glutinosa, Lycium barbarum, Codonopsis pilosula, and Astragalus membranaceus; C. Prepare reference solution; D. Perform high performance liquid chromatography-mass spectrometry analysis on the test sample solution, control medicinal material solution and reference substance solution to obtain the complete components of the Zhanyanming tablets extract.

2. The method according to claim 1, characterized in that The HPLC conditions are: A Thermo 250*4.6mm*5μm column was used, 0.1% formic acid aqueous solution was used as aqueous phase A, and mass spectrometry-grade acetonitrile was used as organic phase B. The gradient elution was as follows: The flow rate was 1.0 mL / min and the column temperature was 35°C.

3. The method according to claim 1, characterized in that The mass spectrometry conditions were as follows: electrospray ionization source, positive and negative ion mode detection, in positive ion mode, the spray voltage was set to 5500 V; in negative ion mode, it was -4500 V, the spray pressure was set to 55 psi, the auxiliary heating pressure was 50 psi, the ion source temperature was maintained at 550°C, the collision voltage was 30 V, the declustering voltage was 80 V, the air curtain pressure was 35 psi, the collider pressure was 10 psi, the scan range covered m / z 50-1500, nitrogen was used as the spray gas and auxiliary gas, and The software collects data in Information Dependent Acquisition (IDA) mode.

4. The method according to claim 1, wherein The test solution is prepared as follows: Weigh 0.5 g of the extract of Zhanyanming tablets, dissolve it in a 25 mL volumetric flask, make up to volume with 75% methanol solution and weigh it, ultrasonicate for 30 min, add to the original weight, and filter through a 0.45 μm microporous filter membrane to obtain the product.

5. The method according to claim 1, characterized in that The preparation method of the control medicinal material solution is as follows: Weigh 6.1 g of Cuscuta seeds, 6.1 g of Rehmannia roots, 4.8 g of Lycium barbarum, 4.8 g of Codonopsis pilosula, and 4.8 g of Astragalus membranaceus, soak them in 100 mL of boiling water for 20 minutes each, and decoct them for 1 hour. Decoction them twice in total, combine the filtrates, concentrate them to 5 mL, and filter them through a 0.45 μm microporous membrane.

6. The method according to claim 1, characterized in that The preparation method of the reference solution is as follows: Weigh 5 mg of reference substance into a volumetric flask, add 25 mL of methanol to each flask and dilute to volume. Dissolve until clear and transparent to obtain a 200 μg / mL reference substance mother solution. Take 5 μL of each reference substance mother solution and mix them. Add methanol to 200 μL and mix evenly.

7. The method according to claim 1, characterized in that The full component analysis is as follows:

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