Inspection method of orifice-opening and deafness-removing pill medicine

Abstract

The invention provides an orifice-opening and deafness-removing pill drug detection method, and particularly relates to the technical field of drug detection. Comprising the following steps: S1, applying UPLC-Q-Exactive Orbitrap / MS to deeply analyze chemical components in the compound traditional Chinese medicine orifice-opening and deafness-removing pills and drug-containing serum of rats of the compound traditional Chinese medicine orifice-opening and deafness-removing pills; s2, comparing the compound components screened in the step S1 with the chemical components identified in the serum in combination with a result of network pharmacology speculation, and determining main drug effect substance components; s3, measuring and confirming the content of the pharmacodynamic substance components determined in the step S2 in the compound by adopting an HPLC (High Performance Liquid Chromatography) method; and S4, comparing the main pharmacodynamic substance components screened in the step S2 with the content components in the compound measured in the step S3 so as to carry out drug inspection. According to the experiment, an HPLC method is adopted for determining the content of main pharmacodynamic substance components in a compound during orifice-opening and deafness-removing pill administration for rats, and the method has a guiding effect on guaranteeing the curative effect and improving the drug quality when being used for drug inspection.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and specifically relates to a drug inspection method for Tongqiao Erlong Pills. Background Art

[0002] Tongqiao Erlong Pills is a traditional Chinese medicine preparation, originating from the famous ancient prescription "Xuanming Lun Fang" written by Liu Wansu in the Jin Dynasty. It belongs to the national essential drugs in the category of ear disease medications and is a Class A drug covered by the national medical insurance. Tongqiao Erlong Pills produced by Hebei Yongfeng Pharmaceutical Co., Ltd., with the approval number Z13020411, contains 12 Chinese herbal medicine ingredients: Bupleuri Radix, Gentianae Radix, Aloe, Rhei Radix Et Rhizoma Preparata, Scutellariae Radix, Indigo Naturalis, Arisaematis Rhizoma (processed with alum), Aucklandiae Radix, Citri Reticulatae Viride Pericarpium (processed with vinegar), Citri Reticulatae Pericarpium, Angelicae Sinensis Radix, Gardeniae Fructus (processed with ginger). This drug has the effects of clearing liver fire, soothing the liver and regulating qi, drying dampness and resolving phlegm, and dredging the orifices and moistening the stool. Currently, it is mainly used clinically for dizziness, tinnitus, earache at the bottom of the ear, bitter taste in the mouth and pain in the hypochondrium, constipation caused by dampness-heat in the liver and gallbladder and hyperactivity of liver-yang, and has good curative effects on various acute ear diseases and vertigo.

[0003] With the development of the modernization of traditional Chinese medicine, the quality control of drugs has become a key link to ensure their efficacy and safety. However, traditional Chinese medicine inspection methods often have problems such as low sensitivity, poor specificity, and cumbersome operations, making it difficult to meet the requirements of modern drug quality control. Especially for compound traditional Chinese medicine preparations, due to their complex ingredients, existing inspection methods often cannot comprehensively and accurately reflect the quality of drugs. Therefore, developing a highly efficient, sensitive, and specific drug inspection method for Tongqiao Erlong Pills has important practical significance. 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 inspection method, the safety of patients' medication can be better guaranteed, and the international competitiveness of traditional Chinese medicine preparations can be enhanced. This patent application aims to provide a drug inspection method for Tongqiao Erlong Pills, which can achieve rapid and accurate detection of the main active ingredients in the drug by optimizing the detection conditions and introducing advanced detection technologies, thereby providing a reliable technical guarantee for the quality control of Tongqiao Erlong Pills. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to propose a drug inspection method for Tongqiao Erlong Pills in view of the deficiencies of the above-mentioned prior art, and specifically adopt the following technical solutions:

[0005] The drug inspection method for Tongqiao Erlong Pills includes the following steps:

[0006] S1. Apply UPLC-Q-Exactive Orbitrap / MS to deeply analyze the chemical components in the compound traditional Chinese medicine Tongqiao Erlong Pills and its rat drug-containing serum;

[0007] S2. Compare the compound components screened in step S1 with the chemical components identified in the serum based on the results speculated by network pharmacology to determine the main active ingredient components.

[0008] S3. Use HPLC method to determine and confirm the content of the active ingredient components determined in step S2 in the compound.

[0009] S4. Compare the main active ingredient components screened in step S2 with the content components measured in the compound in step S3 for drug inspection.

[0010] Preferably, the specific steps of step S1 are as follows:

[0011] S1.1. Prepare the reference substance solution.

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

[0013] S1.3. Prepare the serum sample containing the drug: Randomly divide 6 male SD rats into a blank control group and a Tongqiao Erlong Pills administration group, with 3 rats in each group. The Tongqiao Erlong Pills administration group is given 2 ml of the concentrated solution of Tongqiao Erlong Pills by gavage. After 1 h of gavage, anesthetize with ether, take about 5 mL of abdominal aortic blood, centrifuge at 12000 r / min for 15 min to collect the serum, precipitate the protein with acetonitrile, filter through a 0.22 μm microporous membrane, and store it in a -80°C freezer for standby.

[0014] S1.4. Analyze the components of Tongqiao Erlong Pills and the administered serum by reverse-phase chromatography.

[0015] Preferably, in step S1.4: Through the analysis of reference substances and cleavage rules, 115 components are identified or inferred from the compound, including 2 alkaloid components, 47 flavonoids and their glycosides components, 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, the specific steps of step S2 are as follows:

[0017] S2.1. Screen the target proteins of the compounds entering the blood. Among the 64 components entering the blood, 38 components have clear target proteins, which can affect 258 target proteins.

[0018] S2.2. Analyze the target proteins related to the functions and indications.

[0019] S2.3. Confirmation of the main components related to the functions and indications of Tongqiao Erlong Pills, and it is found that a total of 28 compounds can be preliminarily confirmed as compounds related to the pharmacodynamic material basis.

[0020] Preferably, the 28 compounds screened out in step S2.3 are emodin, rhein, chlorogenic acid, apigenin, nobiletin, hesperidin, poncirin, vitexin, tangeretin, sinensetin, naringenin, 4',5,7-trimethoxyflavone, iristectorigenin B, cyanidin 3-O-glucoside, hesperetin, diosmetin, hyperin, daidzin, quercetin, luteolin, wogonin, beta-D-genistin, kaempferol, vitexicarpin, daidzein, aloin A, costunolic acid, senkyunolide A.

[0021] Preferably, the main pharmacodynamic material basis components that enter the blood screened out in step S2.1 are baicalin, wogonoside, geniposide, hesperidin, luteolin, apigenin.

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

[0023] Preferably, step S1.4 is specifically to establish a self-built database of the chemical components of Tongqiao Erlong Pills, and according to the quasi-molecular ions and secondary fragment information provided by high-resolution mass spectrometry, perform data analysis, and through methods such as comparison with reference substances, retrieval of the self-built database, and retrieval of the online databases Chemical Book, HMDB (http: / / www.hmdb.ca) and Reaxys (https: / / www.reaxys.com / ), speculate and identify each chromatographic peak.

[0024] More preferably, S2.1 is specifically as follows: Use Swiss TargetPredict (http: / / tcmspw.com / ) and TCMSP to predict the action targets of chemical components respectively. For the targets with "validated" retained in the prediction results of TCMSP, and for the prediction results of Swiss TargetPredict, select the targets with a similarity probability value (probability value ≥ 0.5). Further obtain the standardized names of the obtained targets in the Uniport (https: / / www.uniprot.org / ) database, and finally integrate and establish a potential active ingredient-target database.

[0025] Further preferably, in S2.2, specifically, the DisGeNET database is used to import the targets affected by the compound into the database, analyze the associated diseases, screen the main diseases of inflammation, pain, and dizziness, and analyze the targets related to the functions and indications that can be affected by the blood components.

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

[0027] In this experiment, the HPLC method was used to determine the content of the main pharmacodynamic substances of Tongqiao Erlong Pills in the compound in rats. Except for the reference substances of baicalin and wogonoside that could not be covered due to low solubility, the remaining components had good linear relationships in the appropriate injection volume range, and the specificity and precision of the method were good.

[0028] Among the 7 pharmacodynamic components, geniposide, hesperidin, luteolin, apigenin, baicalin, and wogonoside are the basic components of the main pharmacodynamic substances, and gentiopicroside has a relatively high content and is the component for content determination in the current quality standard. Using this method for drug inspection has a guiding role in ensuring the efficacy and improving the drug quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] For ease of explanation, the present invention is described in detail by the following specific embodiments and the accompanying drawings.

[0030] Figure 1 It is the TIC diagram of the TQELW compound. Among them, the upper one is the positive ion mode, and the lower one is the negative ion mode.

[0031] Figure 2 It is the TIC diagram of the drug-containing serum. Among them, the upper one is the positive ion mode, and the lower one is the negative ion mode.

[0032] Figure 3 It is the control chromatogram of the Tongqiao Erlong Pills sample and the mixed reference substances in Example 3. Among them, the upper one is the Tongqiao Erlong Pills sample, and the lower one is the mixed reference substances. The values shown in the figure are gentiopicroside, geniposide, hesperidin, wogonoside, baicalin, luteolin, and apigenin in sequence. DETAILED DESCRIPTION OF THE INVENTION

[0033] The following are specific embodiments of the present invention in combination with the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments; in the following description, providing specific details such as specific configurations is only to help fully understand the embodiments of the present invention. Therefore, those skilled in the art should clearly understand that various changes and modifications can be made to the embodiments described here without departing from the scope and spirit of the present invention.

[0034] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0035] The materials, practices, and experimental equipment involved in the embodiments of the present invention, unless otherwise specified, all comply with commercially available products in the relevant chemical engineering and biotechnology fields.

[0036] Example 1 Analysis of the Compound Prescription and Blood-Entering Components of Tongqiao Erlong Pills Based on UPLC-Q-TOF-MS

[0037] UPLC-Q-Exactive Orbitrap / MS was used to deeply analyze the chemical components of the compound traditional Chinese medicine Tongqiao Erlong Pills and its drug-containing serum in rats. Hypersil GOLD C 18 column (150 mm × 4.6 mm, 1.9 μm) was used to collect the mass spectrometry data of the two samples in positive and negative ion modes respectively. The mass spectrometry data was analyzed by methods such as self-built database, HMDB database, Reaxys database, and comparison with reference substances to achieve the analysis and identification of the chemical components of the samples. A total of 115 compounds were analyzed and identified, including 2 alkaloid components, 47 flavonoid and glycoside components, 6 amino acid components, 15 phenylpropanoid components, 14 organic acid components, and 31 other components.

[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 Substance Solution

[0047] Weigh 10 mg of each reference substance accurately into a 10 mL volumetric flask, add 50% methanol and dissolve it by ultrasonic wave and dilute to the scale. Accurately pipette 100 μL of each reference substance solution into the same 10 mL volumetric flask, add 50% methanol to make up the volume and mix evenly to obtain the reference substance 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, crush it, and pass through a 40-mesh sieve. Weigh 0.5 g of the powder accurately into a stoppered conical flask, add 25 ml of 50% methanol, extract it by ultrasonic wave for 30 min, let it cool, and stand overnight. Take the supernatant and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0050] Preparation of administration solution: Take the above-mentioned medicinal powder, ultrasonically extract it with 15 times of 50% methanol for 30 min, filter and concentrate it, and convert the crude drug amount to 0.4 g / ml.

[0051] 2.3 Preparation of serum samples containing drugs

[0052] Six male SD rats were randomly divided into a blank control group and a Tongqiao Erlong Pills administration group, with 3 rats in each group. The Tongqiao Erlong Pills administration group was given 2 ml of the concentrated solution of Tongqiao Erlong Pills by gavage (the calculated dosage is 0.8 g / 100 g body weight, equivalent to 40 times the clinical dosage), and the blank control group was given 2 mL / 100 g body weight of normal saline by gavage. After 1 h of gavage, the rats were anesthetized with ether, about 5 mL of abdominal aortic blood was taken, centrifuged at 12000 r / min for 15 min to collect serum, the protein was precipitated with acetonitrile, 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] Compound: 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] Serum containing drugs: 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: positive and negative ions simultaneously; Spray voltage: 3.5 kV (positive source), 3.2 kV (negative source), sheath gas, auxiliary gas and purge gas are 50.0, 13.0 and 3.0 μL / min respectively. Capillary temperature: 250 °C; Heater temperature: 400 °C. Scanning mode: Full MS / dd-MS2 mode, mass spectrometry scanning range: 100 - 1500 m / z, scanning speed: 1000 Da / s; Positive and negative ions are collected simultaneously, dynamic deduction is 10 s, collision gas is high-purity nitrogen, spray gas is nitrogen, and it is used for spray-stabilized high-energy collision dissociation (HCD) cell. Number of ions filling the C-Trap: 1e6, maximum injection time: 30 ms. Normalized collision energy (NCE) is 30, 40 and 50 eV; Isolation window (IW) is set to 3; Loop is set to 2.

[0058] 3 Results

[0059] Search the database to establish a self-built database of the chemical components of Tongqiao Erlong Pills. According to the quasi-molecular ions and secondary fragment information provided by high-resolution mass spectrometry, data analysis is carried out. Through methods such as comparison with reference substances, retrieval of the self-built database, and retrieval of the online databases Chemical Book, HMDB (http: / / www.hmdb.ca) and Reaxys (https: / / www.reaxys.com / ), each chromatographic peak is speculated and identified.

[0060] Identification of compounds by reverse-phase chromatography

[0061] Reverse-phase chromatography is used to analyze the components of Tongqiao Erlong Pills and the administered serum. The TIC diagrams of Tongqiao Erlong Pills and the serum samples containing the drug are collected by Q-TOF-MS, and are collected in positive ion mode and negative ion mode, as shown in Figure 1 、 Figure 2 。 By analyzing the reference substances and fragmentation rules and referring to the literature, 115 components are identified or inferred from the compound, including 2 alkaloid components, 47 flavonoid and glycoside components, 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. The compound list is shown in Table 3.

[0062] Table 3 Identification of compounds in the serum containing Tongqiao Erlong Pills under positive and negative ion modes based on UPLC-Q-TOF-MS

[0063]

[0064]

[0065]

[0066] Example 2 Network Pharmacology Prediction Analysis of the Blood-Entering Components of Tongqiao Erlong Pills and Screening and Confirmation of the Components for Content Determination

[0067] Perform network pharmacology research on the 64 chemical components identified or inferred in the serum screened in Example 1 to determine the main pharmacodynamic material basis and guide the screening of the content determination indicators in the quality standard.

[0068] 1 Screening of Blood-Entering Compound Targets

[0069] Use Swiss TargetPredict (http: / / tcmspw.com / ) [1] and TCMSP to predict the action targets of chemical components. Retain the "validated" targets for the prediction results of TCMSP, and select the targets with a similarity probability value (probability value ≥ 0.5) [2] for the prediction results of Swiss TargetPredict. Further obtain the standardized names of the obtained targets on the Uniport (https: / / www.uniprot.org / ) [3] database, and finally integrate and establish a potential active ingredient-target database.

[0070] 2 Analysis of Targets Related to the Functions and Indications

[0071] Use the DisGeNET database to import the targets affected by the compounds into the database, analyze the associated diseases, screen the 4 main diseases of inflammation, pain, vertigo, and depression, and analyze the targets related to the functions and indications that the blood-entering components can affect.

[0072] 3 Confirmation of the Main Components Related to the Functions and Indications of Tongqiao Erlong Pills

[0073] According to the results of the target analysis related to the functions and indications, pair them with the compounds in the active ingredient-target database to determine the main components related to the pharmacodynamic material basis.

[0074] Results:

[0075] 1 Screening of Blood-Entering Compound Targets

[0076] Among the 64 blood-entering components, 38 components have clear action targets and can affect 258 target proteins.

[0077] Table 4 Chemical Composition Table of 38 Blood-Entering Components

[0078]

[0079] Analysis of Targets Related to the Functions and 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, MAPK3

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

[0083] Among the targets that the blood components can act on, there are 8 targets that are related to both analgesic and anti-inflammatory effects: IL10, NOS2, PTGS2, TNF, IL1A, IL6, IL1B, CCL2.

[0084] 3 Confirmation of the Main Components Related to the Functions and Indications of Tongqiao Erlong Pills

[0085] Through database analysis, it is found that a total of 28 compounds can be preliminarily confirmed as the main compounds related to the pharmacodynamic material basis. The main information is shown in Table 5. These components can be used to guide the research on the product quality standard. According to the content of the components in the product, a content determination method can be established for quality control, which can play a role in ensuring the drug efficacy.

[0086] Table 5 Information Table of the Main Compounds Related to the Pharmacodynamic Material Basis

[0087]

[0088]

[0089] Example 3 Quality Inspection of the Tested Drugs of Tongqiao Erlong Pills

[0090] 1 Experimental Reagents

[0091] Table 6 Reagents

[0092]

[0093] Preparation of Solution 2

[0094] Preparation of reference solution: Accurately weigh 7 reference substances, weigh accurately, place them in 25-ml volumetric flasks respectively, dissolve with an appropriate amount of methanol and make up to the mark, shake well. Pipette 1 ml of each solution and transfer it to a 10-ml volumetric flask, make up to the mark with methanol, shake well to obtain the mother liquor of the mixed reference substances. The content of each component per milliliter is as follows: apigenin 81.6 μg, hesperidin 81.6 μg, wogonoside 8.7 μg, luteolin 66.0 μg, baicalin 14.7 μg, gentiopicroside 73.6 μg, gardenoside 75.6 μg.

[0095] Preparation of test solution: Accurately weigh 0.9924 g of Tongqiao Erlong Pills (after removing the coating) in a stoppered conical flask, accurately add 50 ml of methanol, weigh, ultrasonically extract for 30 min, cool to room temperature, stand overnight, make up the weight, filter the supernatant through a 0.22-μm filter membrane for use.

[0096] 3 Chromatographic Conditions and System Suitability Test

[0097] Chromatographic conditions: waters Symmetry C18 chromatographic column; gradient elution with acetonitrile - 0.1% phosphoric acid aqueous solution (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] Inject 10 μL of the reference solution and 10 μL of the test solution into the high-performance liquid chromatograph in sequence, record the chromatogram, and the resolution R between peaks > 1.5.

[0099] 4 Determination of Reference Chromatogram

[0100] Accurately pipette 10 μL of the mixed reference solution under 3.2 and 10 μL of the test solution of Tongqiao Erlong Pills into the high-performance liquid chromatograph, record the chromatogram, and the reference chromatogram is shown in Figure 3 .

[0101] 5 Linear Relationship

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

[0103] 6 Precision

[0104] Take 10 μL of the reference solution and inject it into the high performance liquid chromatograph. Inject continuously for 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 in item 2.2 and 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 plate number

[0110] Analysis: The theoretical plate number calculated according to the hesperidin peak is 3620.

[0111] Result: The theoretical plate number calculated according to the hesperidin peak is greater than 2000.

[0112] Resolution

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

[0114] Result: The baseline separation of each component peak is good (R > 1.5).

[0115] Linearity

[0116] Take 2.0, 5.0, 10.0, 20.0, 30.0, 50.0 μL of the mixed reference solution in item "2.2" and inject them respectively to obtain a series of different injection volumes. Plot the standard curve with the peak area against the injection volume. The abscissa (X) is the peak area, and the ordinate (Y) is the injection volume (μg). The equation and linear range are shown in Table 7.

[0117] Table 7 Record table of standard curve results

[0118]

[0119]

[0120] Precision test

[0121] The RSD values 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 substance.

[0122] Table 8 Record Table of the Precision Test Results of the Injection of the Mixed Reference Substance

[0123]

[0124]

[0125] For the content determination results, 3 batches of test samples were prepared in parallel, and the contents of each component are shown in Table 9.

[0126] Table 9 Content Determination Results of 7 Pharmacodynamic Components in Tongqiao Erlong Pills

[0127]

[0128] In this experiment, HPLC was used to determine the contents of the pharmacodynamic components of Tongqiao Erlong Pills in rats after administration in the compound. Except for the reference substances of baicalin and wogonoside, which could not be covered due to low solubility, the remaining components had good linear relationships in the appropriate injection amount range. The specificity and precision of the method were good, and it was applicable to the content determination of 7 pharmacodynamic components in Tongqiao Erlong Pills. Using this method, the samples of commercially available products were determined, and the average contents of each component were 7.53 mg / g for gentiopicroside, 6.59 mg / g for geniposide, 2.16 mg / g for hesperidin, 10.39 mg / g for baicalin, 7.83 mg / g for wogonoside, 5.45 mg / g for luteolin, and 0.36 mg / g for apigenin respectively.

[0129] Combined with Examples 1 and 2, it can be seen that among these 7 pharmacodynamic components, baicalin, wogonoside, geniposide, hesperidin, luteolin, and apigenin are the main pharmacodynamic substance basis components, and gentiopicroside has a relatively high content and is the component for content determination in the current quality standard. Using this method for drug inspection has a guiding role in ensuring the efficacy and improving the drug quality.

Claims

1. A drug testing method for Tongqiao Erlong Pills, characterized in that: The steps include: S1. UPLC-Q-Exactive Orbitrap / MS was used to deeply analyze the chemical components of the compound Chinese medicine Tongqiao Erlong Pills and its drug-containing serum in rats; S2. Based on the results of network pharmacology inference, the compound ingredients screened out in step S1 and the chemical components identified in the serum are compared to determine the main active substance components; S3, using HPLC to determine and confirm the content of the active substance component determined in step S2 in the compound; S4. Compare the main active substance components screened out in step S2 with the content components in the compound measured in step S3 to conduct drug inspection.

2. The drug testing method for Tongqiao Erlong Pills according to claim 1, characterized in that: The step S1 is specifically as follows: S1.1, prepare reference solution; S1.2, prepare the test solution: take an appropriate amount of 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 bottle, add 25ml of 50% methanol, ultrasonically extract 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: 6 male SD rats were randomly divided into a blank control group and a Tongqiao Erlong Pills administration group, with 3 rats in each group. The Tongqiao Erlong Pills administration group was intragastrically administered with 2 ml of Tongqiao Erlong Pills concentrated solution. One hour after intragastrically administering, ether anesthesia was performed, and about 5 ml of abdominal aorta blood was collected. Serum was collected by centrifugation at 12000 r / min for 15 min, and protein was precipitated with acetonitrile, filtered with a 0.22 μm microporous membrane, and stored in a -80°C freezer for later use. S1.

4. Reverse phase chromatography was used to analyze the components of Tongqiao Erlong Pills and the administered serum.

3. The drug testing method of Tongqiao Erlong Pills according to claim 2, characterized in that: In step S1.4, 115 components were identified or inferred from the compound through reference substances and fragmentation rule analysis, including 2 alkaloid components, 47 flavonoids and their glycoside components, 6 amino acid components, 15 phenylpropanoid components, 14 organic acid components and 31 other components; 64 components were 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.

4. The drug testing method for Tongqiao Erlong Pills according to claim 3, characterized in that: The step S2 is specifically as follows: S2.

1. Target screening of blood-entering compounds: 38 of the 64 blood-entering components have clear targets, affecting 258 target proteins; S2.2, analysis of functional and therapeutic targets; S2.

3. The main ingredients related to the functions and indications of Tongqiao Erlong Pills were confirmed, and it was found that a total of 28 compounds could be preliminarily confirmed as compounds related to the material basis of the efficacy.

5. The drug testing method for Tongqiao Erlong Pills according to claim 4, characterized in that: The 28 compounds screened out in step S2.3 are rhein, rhein, chlorogenic acid, apigenin, nobiletin, hesperidin, citrus glycosides, vitexin, hesperetin, sweet orange flavonoids, naringenin, 4',5,7-trimethoxyflavone, irisin B, cyanidin, hesperetin, diosmetin, hyperoside, daidzein, quercetin, luteolin, baicalin, beta-D-genistein, kaempferol, vitexin, glycitein, aloin A, costus acid, and ligusticum chuanxiong lactone A.

6. The drug testing method for Tongqiao Erlong Pills according to claim 4, characterized in that: The main active substance basic components that enter the blood screened out in step S2.1 are baicalin, wogonin, gardenia glycoside, hesperidin, luteolin, and apigenin.

7. The drug testing method for Tongqiao Erlong Pills according to claim 3 or 4, characterized in that: The ingredients identified in the compound in step S1.4 include gentiopicroside, which can be used as a content determination component in the quality standard.

8. The drug testing method for Tongqiao Erlong Pills according to claim 3 or 4, characterized in that: The step S1.4 is specifically as follows: establishing a self-built database of the chemical components of Tongqiao Erlong Pills, 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 substances, self-built database retrieval, and online database ChemicalBook, HMDB and Reaxys retrieval methods.

9. The drug testing method for Tongqiao Erlong Pills according to claim 4, characterized in that: The step S2.1 is specifically as follows: using Swiss TargetPredict and TCMSP to predict the targets of chemical components respectively, retaining the "validated" targets for the TCMSP prediction results, selecting the targets with similarity probability value ≥ 0.5 for the Swiss TargetPredict prediction results, further obtaining standardized names for the obtained targets on the Uniport database, and finally integrating and establishing a potential active ingredient-target database.

10. The drug testing method for Tongqiao Erlong Pills according to claim 4, characterized in that: The step S2.2 is specifically as follows: using the DisGeNET database, the targets affected by the compounds are imported into the database, the diseases associated with the compounds are analyzed, inflammation, pain, and dizziness are screened as the main diseases, and the targets related to the functional indications that can be affected by the blood components are analyzed.

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