Drug inspection method for tongqiao erding pill

Abstract

The application provides a method for detecting the Tongqiao Erlong pill, and particularly relates to the technical field of drug detection. The method comprises the following steps: S1, applying UPLC-Q-Exactive Orbitrap / MS to deeply analyze the chemical components in the Tongqiao Erlong pill and the rat serum containing the medicine; S2, comparing the chemical components in the serum identified in step S1 with the components screened out by network pharmacology, so as to determine the main pharmacodynamic material components; S3, using the HPLC method to determine and confirm the content of the pharmacodynamic material components determined in step S2 in the compound; and S4, comparing the main pharmacodynamic material components screened out in step S2 with the content components in the compound measured in step S3, so as to perform drug detection. In the experiment, the HPLC method is used to determine the content of the main pharmacodynamic material components of the Tongqiao Erlong pill in the compound, and the method is used for drug detection, which can guide the guarantee of curative effect and the improvement of drug quality.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical detection, in particular to a method for testing Tongqiao Erlong Pills. Background Art

[0002] Tongqiao Erlong Pills is a traditional Chinese medicine preparation derived from the ancient prescription of famous physician Liu Wansu in the Jin Dynasty recorded in *Xuanming Lun Fang (Discourses on Prescription by Xuanming)*. It is a national essential medicine for ear diseases and a national Class A medicine covered by medical insurance. The Tongqiao Erlong Pills produced by Hebei Yongfeng Pharmaceutical Co., Ltd. with the approval number Z13020411 contains 12 traditional Chinese medicinal ingredients: Bupleuri Radix, Gentianae Radix et Rhizoma, Aloe, Rhei Radix et Rhizoma Praeparata, Scutellariae Radix, Indigo Naturalis, Arisaematis Rhizoma (alum-processed), Aucklandiae Radix, Citri Reticulatae Viride Pericarpium (vinegar-processed), Citri Reticulatae Pericarpium, Angelicae Sinensis Radix, and Gardeniae Fructus (ginger-processed). The medicine has the effects of clearing liver fire, soothing liver qi, drying dampness and resolving phlegm, opening orifices and moistening intestines. Currently, it is clinically mainly used for dizziness, deafness and tinnitus, swelling and pain in the ear base, bitter mouth and hypochondriac pain, and constipation caused by dampness-heat in liver and gallbladder and hyperactivity of liver yang, and has good curative effect on various acute ear diseases and vertigo.

[0003] With the development of modernization of traditional Chinese medicine, drug quality control has become a key link to ensure its efficacy and safety. However, traditional traditional Chinese medicine testing methods often have problems such as low sensitivity, poor specificity and cumbersome operation, which are difficult to meet the requirements of modern drug quality control. Especially for compound traditional Chinese medicine preparations, due to their complex components, existing testing methods often cannot fully and accurately reflect the quality of drugs. Therefore, it is of great practical significance to develop an efficient, sensitive and highly specific method for testing Tongqiao Erlong Pills. This method can not only ensure the stability and consistency of drug quality, but also provide technical support for the modern production and quality control of traditional Chinese medicine preparations. By establishing a scientific testing method, it can better guarantee the medication safety of patients and enhance the international competitiveness of traditional Chinese medicine preparations. The present patent application aims to provide a method for testing Tongqiao Erlong Pills, which realizes rapid and accurate detection of main active components in the drug by optimizing detection conditions and introducing advanced detection technologies, so as to provide reliable technical guarantee for the quality control of Tongqiao Erlong Pills. Summary of the Invention

[0004] Aiming at the deficiencies in the above prior art, the technical problem to be solved by the present invention is to provide a method for testing Tongqiao Erlong Pills, and specifically adopts the following technical solution:

[0005] A method for testing Tongqiao Erlong Pills comprises the following steps:

[0006] S1, using UPLC-Q-Exactive Orbitrap / MS to deeply analyze the chemical components of the compound traditional Chinese medicine Tongqiao Erlong Pills and the drug-containing serum of rats administered with Tongqiao Erlong Pills;

[0007] S2. Based on the results of network pharmacology, the compound components screened in step S1 and the chemical components identified in serum are compared to determine the main pharmacodynamic components.

[0008] S3. The content of the active pharmaceutical ingredients identified in step S2 in the compound was determined and confirmed by HPLC.

[0009] S4. The main active pharmaceutical ingredients screened in step S2 are compared with the content of the ingredients in the compound measured in step S3 for drug testing.

[0010] Preferably, step S1 specifically includes:

[0011] S1.1, Prepare the reference solution;

[0012] S1.2 Preparation of test solution: Take an appropriate amount of commercially available Tongqiao Erlong Pills, scrape off the white coating, pulverize, and pass through a 40-mesh sieve. Accurately weigh 0.5g of powder into a stoppered conical flask, add 25ml of 50% methanol, extract by sonication for 30min, cool, let stand overnight, and filter the supernatant through a 0.22μm filter membrane to obtain the test solution.

[0013] S1.3 Preparation of drug-containing serum samples: Six male SD rats were randomly divided into a blank control group and a Tongqiao Erlongwan administration group, with three rats in each group. The Tongqiao Erlongwan administration group was given 2 ml of concentrated Tongqiao Erlongwan solution by gavage. One hour after gavage, the rats were anesthetized with ether, and about 5 mL of blood was collected from the abdominal aorta. The serum was collected by centrifugation at 12000 r / min for 15 min. The protein was precipitated with acetonitrile, filtered through a 0.22 μm microporous membrane, and stored in a freezer at -80℃ for later use.

[0014] S1.4. Reversed-phase chromatography was used to analyze the components of Tongqiao Erlong Pill and the serum after administration.

[0015] Preferably, in step S1.4: through reference standards and analysis of lysis patterns, 115 components are identified or inferred from the compound, including 2 alkaloid components, 47 flavonoids and their glycosides, 6 amino acid components, 15 phenylpropanoid components, 14 organic acid components, and 31 other components; 64 components are identified or inferred from the administered serum, including 40 flavonoid components, 4 triterpenoid components, 4 lactone components, 7 anthraquinone components, 2 aromatic acid components, and 7 other components.

[0016] Preferably, step S2 specifically comprises:

[0017] S2.1 Screening of targets for compounds entering the bloodstream: Among 64 components entering the bloodstream, 38 components have clear targets and can affect 258 target proteins.

[0018] S2.2 Analysis of target points related to therapeutic indications;

[0019] S2.3. The main components related to the functions and indications of Tongqiao Erlong Pill were identified, and a total of 28 compounds were found to be preliminarily identified as compounds related to the pharmacodynamic material basis.

[0020] Preferably, the 28 compounds screened in step S2.3 are emodin, rhein, chlorogenic acid, apigenin, norihesperidin, hesperidin, limonin, vitexin, citrus peel, sweet orange flavonoids, naringenin, 4',5,7-trimethoxyflavonoids, iris flavonoid B, brown cyanidin, hesperidin, geraniol, hyperoside, daidzein, quercetin, luteolin, baicalin, beta-D-genistein, kaempferol, vitexin, daidzein, aloe-emodin A, costolic acid, and ligustrol A.

[0021] Preferably, the main pharmacodynamic components screened in step S2.1 that enter the bloodstream are baicalin, wogonin, geniposide, hesperidin, luteolin, and apigenin.

[0022] Preferably, the components identified in the compound in step S1.4 include gentiopicrin, which can be used as the content determination component in the quality standard.

[0023] Preferably, step S1.4 specifically involves establishing a self-built database of the chemical components of Tongqiao Erlong Pill, performing data analysis based on the quasi-molecular ion and secondary fragment information provided by high-resolution mass spectrometry, and speculating and identifying each chromatographic peak through comparison with reference standards, retrieval of the self-built database, and retrieval of online databases such as Chemical Book, HMDB (http: / / www.hmdb.ca) and Reaxys (https: / / www.reaxys.com / ).

[0024] More preferably, step S2.1 specifically involves: using Swiss TargetPredict (http: / / tcmspw.com / ) and TCMSP to predict the target sites of chemical components, retaining the "validated" targets from the TCMSP prediction results, selecting the targets with a similarity probability value (probability value ≥ 0.5) from the Swiss TargetPredict prediction results, further obtaining standardized names for the obtained targets from the Uniport (https: / / www.uniprot.org / ) database, and finally integrating and establishing a potential active ingredient-target database.

[0025] More preferably, in step S2.2, the DisGeNET database is used to import the targets affected by the compound into the database, analyze the diseases associated with them, screen for major diseases such as inflammation, pain, and dizziness, and analyze the targets related to the therapeutic functions that can be affected by the blood-entering components.

[0026] Compared with the prior art, the present invention achieves the following technical effects:

[0027] This experiment used HPLC to determine the content of the main active pharmaceutical ingredients in the compound of Tongqiao Erlong Pill administered to rats. Except for baicalin and wogonin reference standards which were not included due to their low solubility, the remaining components showed good linearity within the appropriate injection volume range. The method has good specificity and precision.

[0028] Among the seven active pharmaceutical ingredients, geniposide, hesperidin, luteolin, apigenin, baicalin, and wogonin are the main active pharmaceutical ingredients. Gentianoside is a relatively high-content ingredient and is currently included in the content determination standards. Using this method for drug testing can provide guidance for ensuring efficacy and improving drug quality. Attached Figure Description

[0029] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 The image shows the TQELW compound TIC pattern, where the top represents positive ion mode and the bottom represents negative ion mode.

[0031] Figure 2 The image shows the TIC (Total Ion Conversion) curve of drug-containing serum, with the top showing positive ion mode and the bottom showing negative ion mode.

[0032] Figure 3 This is a comparison chromatogram of the Tongqiao Erlong Pill sample and the mixed control in Example 3. The top part represents the Tongqiao Erlong Pill sample, and the bottom part represents the mixed control. The values ​​shown in the figure are, in order, gentiopicrin, geniposide, hesperidin, baicalin, scutellarin, luteolin, and apigenin. Detailed Implementation

[0033] The following are specific embodiments of the present invention, described in conjunction with the accompanying drawings, to further illustrate the technical solutions of the present invention. However, the present invention is not limited to these embodiments. Specific details, such as particular configurations, are provided in the following description merely to aid in a comprehensive understanding of the embodiments of the present invention. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0035] Unless otherwise specified, the materials, practices, and experimental equipment involved in the embodiments of this invention are all commercially available products in the relevant chemical and biotechnology fields.

[0036] Example 1: Analysis of Tongqiao Erlong Wan Compound and Blood Components Based on UPLC-Q-TOF-MS

[0037] UPLC-Q-Exactive Orbitrap / MS was used to thoroughly analyze the chemical components of the compound traditional Chinese medicine Tongqiao Erlong Wan and its drug-containing rat serum. Hypersil GOLD C was used as the analytical medium. 18 Mass spectrometry data of two samples were acquired using a column (150 mm × 4.6 mm, 1.9 μm) in both positive and negative ion modes. The mass spectrometry data were analyzed using a self-built database, HMDB database, Reaxys database, and reference standard comparison methods to achieve chemical composition analysis and identification of the samples. A total of 115 compounds were analyzed and identified, including 2 alkaloids, 47 flavonoids and their glycosides, 6 amino acids, 15 phenylpropanoids, 14 organic acids, and 31 other compounds.

[0038] 1. Instruments and Materials

[0039] Table 1 Instruments

[0040]

[0041] Table 2 Reagents

[0042]

[0043]

[0044]

[0045] 2 methods

[0046] 2.1 Preparation of reference solution

[0047] Accurately weigh 10 mg of each reference standard into a 10 mL volumetric flask, add 50% methanol to dissolve by sonication and dilute to the mark, accurately pipette 100 μL of each reference standard solution into the same 10 mL volumetric flask, add 50% methanol to dilute to volume and mix well to obtain the reference standard test solution.

[0048] 2.2 Preparation of test solution

[0049] Take an appropriate amount of commercially available Tongqiao Erlong Pills, scrape off the white coating, pulverize, and pass through a 40-mesh sieve. Accurately weigh 0.5g of powder into a stoppered conical flask, add 25ml of 50% methanol, extract by sonication for 30min, cool, and let stand overnight. Filter the supernatant through a 0.22μm filter membrane to obtain the test solution.

[0050] Preparation of administration solution: take the above medicinal powder, perform ultrasonic extraction with 15 times the amount of 50% methanol for 30 min, filter and concentrate to obtain a solution equivalent to 0.4 g of crude drug per ml.

[0051] 2.3 Preparation of drug-containing serum samples

[0052] Six male SD rats were randomly divided into a blank control group and a Tongqiao Erlong Wan administration group, with 3 rats in each group. The Tongqiao Erlong Wan administration group was intragastrically administered 2 ml of concentrated Tongqiao Erlong Wan solution (converted to an administration dose of 0.8 g / 100 g body weight, equivalent to 40 times the clinical dose), and the blank control group was intragastrically administered normal saline at a dose of 2 mL / 100 g body weight. After 1 h of intragastric administration, the rats were anesthetized with ether, and about 5 mL of abdominal aortic blood was collected. The serum was collected by centrifugation at 12000 r / min for 15 min. Protein was precipitated with acetonitrile, and the supernatant was filtered through a 0.22 μm microporous membrane, and stored in a -80°C freezer for later use. All rats were euthanized after blood collection.

[0053] 2.4 Chromatographic conditions

[0054] For the compound prescription: Mobile phase: 0.1% formic acid water (A)-acetonitrile solution (B), gradient elution: 0-2 min, 5% B; 2-13 min, 5%→75% B; 13-25 min, 24%→32% B; 25-63 min, 32%→95% B; 63-63.1 min, 95%→5% B; 63.1-65 min, 5% B; flow rate: 0.3 ml·min-1; column temperature: 30°C; injection volume: 5 μl.

[0055] For drug-containing serum: Mobile phase: 0.1% formic acid water (A)-methanol solution (B), gradient elution: 0-2 min, 5% B; 2-13 min, 5%→24% B; 13-32 min, 75%→95% B; 32-35 min, 95% B; 35-36 min, 95%→5% B; 36-39 min, 5% B; flow rate: 0.3 ml·min-1; column temperature: 30°C; injection volume: 5 μl.

[0056] 2.5 Mass spectrometry parameters

[0057] Ion source: Electrospray ionization (ESI) mode; Detection mode: Simultaneous positive and negative ion collection; Spray voltage: 3.5kV (positive source), 3.2kV ​​(negative source); Sheath gas, auxiliary gas, and purge gas flow rates: 50.0, 13.0, and 3.0 μL / min, respectively. Capillary temperature: 250℃; Heater temperature: 400℃. Scanning mode: Full MS / dd-MS2 mode; Mass spectrometry scan range: 100-1500 m / z; Scan rate: 1000 Da / s; Simultaneous positive and negative ion acquisition with a dynamic subtraction of 10 s; Collision gas: High-purity nitrogen; Spray gas: Nitrogen; Used for spray-stabilized high-energy collisional dissociation (HCD) cells. Number of ions filling the C-Trap: 1e6; Maximum injection time: 30 ms. The normalized collision energy (NCE) is 30, 40, and 50 eV; the isolation window (IW) is set to 3; and the loop is set to 2.

[0058] 3 Results

[0059] A self-built database of the chemical components of Tongqiao Erlong Pill was established by searching databases. Data analysis was performed based on the quasi-molecular ion and secondary fragment information provided by high-resolution mass spectrometry. The chromatographic peaks were inferred and identified through comparison with reference standards, searching the self-built database, and searching online databases such as Chemical Book, HMDB (http: / / www.hmdb.ca), and Reaxys (https: / / www.reaxys.com / ).

[0060] Reversed-phase chromatography for the identification of compounds

[0061] Component analysis of Tongqiao Erlong Pills and drug-containing serum was performed using reversed-phase chromatography. TIC chromatograms of the Tongqiao Erlong Pills and drug-containing serum samples were acquired by Q-TOF-MS, with acquisition performed in both positive and negative ion modes. (See figure) Figure 1 , Figure 2 Through analysis of reference standards and fragmentation patterns, and consulting relevant literature, 115 components were identified or inferred from the compound preparation, including 2 alkaloids, 47 flavonoids and their glycosides, 6 amino acids, 15 phenylpropanoids, 14 organic acids, and 31 other components. 64 components were identified or inferred from the administered serum, including 40 flavonoids, 4 triterpenoids, 4 lactones, 7 anthraquinones, 2 aromatic acids, and 7 other components. A list of compounds is provided in Table 3.

[0062] Table 3. Identification of compounds in TQELW drug-containing serum under positive and negative ion modes based on UPLC-Q-TOF-MS.

[0063]

[0064]

[0065]

[0066] Example 2: Network pharmacological prediction analysis and content determination of components entering the bloodstream of Tongqiao Erlong Pills - screening and confirmation of components.

[0067] Network pharmacology studies were conducted on 64 chemical components identified or inferred in serum from Example 1 to determine the main pharmacodynamic material basis and guide the screening of content determination indicators in quality standards.

[0068] 1. Screening of blood-borne compound targets

[0069] Use Swiss TargetPredict (http: / / tcmspw.com / ) respectively. [1] The targets of chemical composition were predicted using TCMSP. Targets marked as "validated" in the TCMSP prediction results were retained, while the similarity probability value (probability value ≥ 0.5) of the Swiss TargetPredict prediction results was selected. [2] The target points obtained will be further processed on Uniport (https: / / www.uniprot.org / ). [3] Standardized nomenclature is obtained from the database, and finally integrated and established as a potential active ingredient-target database.

[0070] 2. Analysis of target points related to therapeutic indications

[0071] Using the DisGeNET database, the targets affected by the compounds were imported into the database to analyze the associated diseases. Four major diseases were screened: inflammation, pain, dizziness, and depression. The targets related to the therapeutic indications that the blood-entered components could affect were analyzed.

[0072] 3. Confirmation of the main ingredients related to the indications and functions of Tongqiao Erlong Pill.

[0073] Based on the target analysis results related to the indications and functions, and by comparing them with compounds in the active ingredient-target database, the main components related to the pharmacodynamic material basis are identified.

[0074] result:

[0075] 1. Screening of blood-borne compound targets

[0076] Of the 64 components that enter the bloodstream, 38 have clearly defined targets and can affect 258 target proteins.

[0077] Table 438 Chemical Composition Table of Blood-Entering Components

[0078]

[0079] 2. Analysis of target points related to therapeutic indications

[0080] There are 29 inflammation-related targets: CRP, CSF2, AHR, PTGS2, MPO, TNF, CXCL2, ICAM1, CCL2, AKT1, HMOX1, TIMP1, IL10, PTPN1, TGFB1, NOS2, MMP2, IL13, ADIPOQ, MMP9, SOD1, VEGFA, IL1A, IL6, IFNG, IL1B, PPARG, PPARA, PTGES

[0081] There are 20 pain-related targets: IL10, PRKCG, NOS2, PRKCE, INSR, MMP3, FOS, PTGS2, TNF, PTGS1, IL1A, THBD, IL6, MAPK8, MAPK7, IL1B, CCL2, MAPK1, RAF1, and MAPK3.

[0082] There are 13 targets related to vertigo: ACHE, CHRM1, NOS2, SLC2A1, ABAT, FOS, PTGS2, ADRA1B, SLC6A2, INS, IL6, IL1B, and CAT.

[0083] Among the targets that blood-entering components can act on, there are eight targets that can be associated with analgesia and anti-inflammatory effects: IL10, NOS2, PTGS2, TNF, IL1A, IL6, IL1B, and CCL2.

[0084] 3. Confirmation of the main ingredients related to the indications and functions of Tongqiao Erlong Pill.

[0085] Database analysis revealed 28 compounds that can be preliminarily identified as key compounds related to the pharmacodynamic basis, as shown in Table 5. These components can guide product quality standard research, and content determination methods can be established based on their concentration in products for quality control, thus ensuring drug efficacy.

[0086] Table 5. Information on major compounds related to the pharmacodynamic material basis.

[0087]

[0088]

[0089] Example 3: Quality Inspection of the Test Drug, Tongqiao Erlong Pill

[0090] 1. Reagents used in the experiment

[0091] Table 6 Reagents

[0092]

[0093] 2. Preparation of solution

[0094] Preparation of reference solutions: Accurately weigh seven reference standards and place them separately in 25ml volumetric flasks. Add an appropriate amount of methanol to dissolve and dilute to the mark, shake well, and transfer 1ml of each to a 10ml volumetric flask. Add methanol to the mark and shake well to obtain the mixed reference standard stock solution. The content of each component per milliliter is as follows: apigenin 81.6μg, hesperidin 81.6μg, baicalin 8.7μg, luteolin 66.0μg, baicalin 14.7μg, gentiopicroside 73.6μg, and geniposide 75.6μg.

[0095] Preparation of test solution: Accurately weigh 0.9924 g of Tongqiao Erlong Pill (with coating removed) dry powder into a stoppered conical flask, accurately add 50 ml of methanol, weigh, sonicate for 30 min, cool, let stand overnight, add weight, take the supernatant and filter through a 0.22 μm filter membrane, and set aside for use.

[0096] 3. Chromatographic conditions and system suitability test

[0097] Chromatographic conditions: Waters Symmetry C18 column; acetonitrile-0.1% phosphoric acid aqueous gradient elution (0–5 min, 5% → 15% A; 5–20 min, 15% A → 20% A; 20–40 min, 20% → 40% A; 40–45 min, 40% A → 90% A; 45–55 min, 90% A; 55–56 min, 90% A → 5% A; 56–66 min, 5% A); flow rate 0.8 ml / min; detection wavelength 240 nm; column temperature: 25 °C.

[0098] 10 μL of the reference solution and 10 μL of the test solution were injected sequentially into the high performance liquid chromatograph, and the chromatograms were recorded. The resolution R between each peak was greater than 1.5.

[0099] 4. Spectrum determination

[0100] Accurately pipette 10 μL of the mixed reference solution from section 3.2 and 10 μL of the Tongqiao Erlong Pill test solution into the high-performance liquid chromatograph, record the chromatogram, and refer to the comparison chromatogram. Figure 3 .

[0101] 5. Linear Relationship

[0102] Inject 2.0, 5.0, 10.0, 20.0, 30.0, and 50.0 μL of the mixed reference solution to obtain a series of different injection volumes. Plot a standard curve with peak area and injection volume. The x-axis (X) represents the peak area ( / ml), and the y-axis (Y) represents the injection volume (μg). The correlation coefficient r should be ≥ 0.99.

[0103] 6 Precision

[0104] Inject 10 μL of the reference solution into the high-performance liquid chromatograph, repeat the injection 6 times, record the chromatogram, and calculate the RSD. The RSD value should be ≤2%.

[0105] 7. Content determination

[0106] Take 10 μL of the test solution prepared under section 2.2, inject it into the liquid chromatograph, observe the chromatogram or calculate the content of each target component.

[0107] 8. Results:

[0108] System suitability test

[0109] Theoretical number of plates

[0110] Analysis: The theoretical plate number, calculated based on the hesperidin peak, is 3620.

[0111] Results: The theoretical plate number, calculated based on the hesperidin peak, was greater than 2000.

[0112] Resolution

[0113] Analysis: The chromatogram records the minimum resolution R of the reference solution as 1.56.

[0114] Results: The baselines of each component peak were well separated (R>1.5).

[0115] linear

[0116] Inject 2.0, 5.0, 10.0, 20.0, 30.0, and 50.0 μL of the mixed reference solution under section “2.2” to obtain a series of different injection volumes. Plot a standard curve with peak area and injection volume. The x-axis (X) represents the peak area, and the y-axis (Y) represents the injection volume (μg). The equation and linear range are shown in Table 7.

[0117] Table 7 Standard Curve Results Record Table

[0118]

[0119]

[0120] Precision test

[0121] The RSDs were 1.72%, 1.63%, 1.94%, 1.49%, 1.45%, 1.50%, and 0.72%, respectively, indicating good precision in the injection of the reference standard.

[0122] Table 8 Record of Precision Test Results for Mixed Reference Standard Injection

[0123]

[0124]

[0125] The content determination results were obtained by preparing three batches of test samples in parallel and determining the content of each component, as shown in Table 9.

[0126] Table 9. Results of content determination of 7 active ingredients in Tongqiao Erlong Pills

[0127]

[0128] This experiment used HPLC to determine the content of the active ingredients in Tongqiao Erlong Pills administered to rats. Except for baicalin and wogonin reference standards, which were not included due to their low solubility, the remaining components showed good linearity within a suitable injection range. The method demonstrated good specificity and precision, and is suitable for determining the content of the seven active ingredients in Tongqiao Erlong Pills. Using this method, samples of commercially available products were tested. The average contents of each ingredient were as follows: gentiopicroside 7.53 mg / g, geniposide 6.59 mg / g, hesperidin 2.16 mg / g, baicalin 10.39 mg / g, wogonin 7.83 mg / g, luteolin 5.45 mg / g, and apigenin 0.36 mg / g.

[0129] Based on Examples 1 and 2, it can be seen that among these seven pharmacologically active ingredients, baicalin, wogonin, geniposide, hesperidin, luteolin, and apigenin are the main pharmacologically active basic components, while gentiopicrin is a relatively high-content component that is currently included in the content determination in quality standards. Using this method for drug testing can provide guidance for ensuring efficacy and improving drug quality.

Claims

1. A drug testing method for Tongqiao Erlong Pills, characterized in that, Includes the following steps: S1.1, Prepare the reference solution; S1.2 Preparation of test solution: Take an appropriate amount of commercially available Tongqiao Erlong Pills, scrape off the white coating, crush, pass through a 40-mesh sieve, accurately weigh 0.5g of powder into a stoppered conical flask, add 25ml of 50% methanol, extract by ultrasonication for 30min, cool, let stand overnight, take the supernatant and filter through a 0.22μm filter membrane to obtain the test solution; S1.3 Preparation of drug-containing serum samples: Six male SD rats were randomly divided into a blank control group and a Tongqiao Erlongwan administration group, with three rats in each group. The Tongqiao Erlongwan administration group was given 2 ml of concentrated Tongqiao Erlongwan solution by gavage. One hour after gavage, the rats were anesthetized with ether, and about 5 mL of blood was collected from the abdominal aorta. The serum was collected by centrifugation at 12000 r / min for 15 min. The protein was precipitated with acetonitrile, filtered through a 0.22 μm microporous membrane, and stored in a freezer at -80℃ for later use. S1.

4. Reversed-phase chromatography was used to analyze the components of Tongqiao Erlong Pills and serum samples containing the drug. 115 components were identified or inferred from Tongqiao Erlong Pill, including 2 alkaloids, 47 flavonoids and their glycosides, 6 amino acids, 15 phenylpropanoids, 14 organic acids, and 31 other components; 64 components were identified or inferred from drug-containing serum samples, including 40 flavonoids, 4 triterpenoids, 4 lactones, 7 anthraquinones, 2 aromatic acids, and 7 other components. S2.1 Screening of blood-entering compounds for targets: Among 64 blood-entering components, 38 components have clear targets and can affect 258 target proteins; S2.2 Analysis of target points related to therapeutic indications; S2.

3. The main components related to the functions and indications of Tongqiao Erlong Pill were identified, and a total of 28 compounds were found to be preliminarily identified as compounds related to the pharmacodynamic material basis. S3. The content of the compounds related to the pharmacodynamic material basis identified in step S2.3 in Tongqiao Erlong Pill was determined and confirmed by HPLC. S4. The compounds related to the pharmacodynamic material basis screened in step S2.3 are compared with the components in Tongqiao Erlong Pills measured in step S3 for drug testing. The 28 compounds screened in step S2.3 are emodin, rhein, chlorogenic acid, apigenin, norihesperidin, hesperidin, limonin, vitexin, citrus peel, sweet orange flavonoids, naringenin, 4',5,7-trimethoxyflavonoids, iris flavonoid B, brown cyanidin, hesperidin, geraniol, hyperoside, daidzein, quercetin, luteolin, baicalin, beta-D-genistein, kaempferol, vitexin, daidzein, aloe-emodin A, costolic acid, and ligustrol A. The main pharmacologically active ingredients screened in step S2.1 that enter the bloodstream are baicalin, wogonin, geniposide, hesperidin, luteolin, and apigenin. The chromatographic conditions in step S1.4 are as follows: Tongqiao Erlong Pill: Mobile phase: 0.1% formic acid aqueous solution-acetonitrile solution, gradient elution: 0~2 min, 5% B; 2~13 min, 5%→75% B, 13~25 min, 24%→32% B, 25~63 min, 32%→95% B, 63~63.1 min, 95%→5% B, 63.1~65 min, 5% B; flow rate: 0.3 ml·min⁻¹; column temperature: 30℃; injection volume: 5 μl; Drug-containing serum sample: mobile phase: 0.1% formic acid water-methanol solution, gradient elution: 0~2 min, 5% B; 2~13 min, 5%→24% B, 13~32 min, 75%→95% B, 32~35 min, 95% B, 35~36 min, 95%→5% B, 36~39 min, 5% B; flow rate 0.3 ml·min -1 ; column temperature 30°C; injection volume 5 μl; The mass spectrometry parameters are: Ion source: Electrospray ionization mode; Detection mode: Simultaneous positive and negative ion detection; Spray voltage: 3.5kV positive source, 3.2kV ​​negative source; Sheath gas, auxiliary gas, and purge gas flow rates: 50.0, 13.0, and 3.0µL / min, respectively; Capillary temperature: Heater temperature: The scanning mode was Full MS / dd-MS2, with a mass spectrometry scanning range of 100-1500 m / z and a scanning speed of 1000 Da / s. Simultaneous acquisition of positive and negative ions was used, with a dynamic subtraction time of 10 s. High-purity nitrogen was used as the collision gas and nitrogen as the spray gas. The number of ions filling the C-Trap was 1e6, and the maximum injection time was 30 ms. The normalized collision energies were 30 eV, 40 eV, and 50 eV. The isolation window was set to 3, and the Loop was set to 2.

2. The method for testing the Tongqiao Erlong Pill according to claim 1, characterized in that, The components identified in the Tongqiao Erlong Pill in step S1.4 include gentiopicrin, which can be used as a content determination component in the quality standard.

3. The method for testing the Tongqiao Erlong Pill according to claim 1, characterized in that, Step S1.4 specifically involves: establishing a self-built database of chemical components for Tongqiao Erlong Pills; conducting data analysis based on the quasi-molecular ion and secondary fragment information provided by high-resolution mass spectrometry; and speculating and identifying each chromatographic peak through comparison with reference standards, retrieval of the self-built database, and retrieval of online databases such as Chemical Book, HMDB, and Reaxys.

4. The method for testing the Tongqiao Erlong Pill according to claim 1, characterized in that, Step S2.1 specifically involves: using Swiss TargetPredict and TCMSP to predict the target sites of chemical components, retaining "validated" targets from the TCMSP prediction results, selecting targets with a similarity probability value ≥ 0.5 from the Swiss TargetPredict prediction results, obtaining standardized names for the obtained targets from the Uniport database, and finally integrating and establishing a potential active ingredient-target database.

5. The method for testing the Tongqiao Erlong Pill according to claim 1, characterized in that, Step S2.2 specifically involves: using the DisGeNET database, importing the targets affected by the compound into the database, analyzing the associated diseases, screening for major diseases such as inflammation, pain, and dizziness, and analyzing the targets related to the therapeutic functions that can be affected by the blood-entering components.

Images