Methods for identifying quality markers of traditional Chinese medicine processing

Abstract

The present invention discloses a method for identifying quality markers of Chinese medicine processing. The method comprises the following steps: step A: determining the blood-entering components of Chinese medicine processing products; step B: performing network pharmacology analysis of the blood-entering components of Chinese medicine processing products; step C: comparing the blood-entering components of raw Chinese medicine products with those of processed Chinese medicine products; and step D: identifying the quality markers of processed Chinese medicine processing products. The method for identifying quality markers of Chinese medicine processing of the present invention combines network pharmacology research to determine the primary and secondary relationship of blood-entering components, and determines the quality markers of Chinese medicine processing by comparative analysis of the blood-entering components of processed products and raw products, so as to improve the stability and controllability of the quality of processed Chinese medicine products.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a method for identifying quality markers of traditional Chinese medicine preparation. Background Art

[0002] Chinese medicine processing refers to the process of necessary processing of drugs according to the needs of medical treatment, modulation, and preparation before they are used or made into various dosage forms. It is a unique Chinese medicine pharmaceutical technology in my country. Chinese medicine processing is a traditional pharmaceutical technology and a key point connecting Chinese medicine and Chinese pharmacology. Studies have shown that changes in the chemical composition of Chinese medicine during the processing of Chinese medicine lead to changes in its pharmacological effects. The chemical changes in these substances are the important reasons for the changes in the nature, taste and function of Chinese medicine before and after processing, which also determines the complexity of the control of the Chinese medicine processing process and the formulation of quality standards for processed products. Therefore, the research progress of Chinese medicine processing has been relatively slow, and the quality control research of Chinese medicine processing has always lagged behind the quality control research of raw Chinese medicine.

[0003] The current "quality markers of traditional Chinese medicine" are proposed to integrate multidisciplinary knowledge, propose core quality concepts, and further strengthen the correlation between the effectiveness of traditional Chinese medicine, material basis, and quality control characteristic components of traditional Chinese medicine, which is conducive to the establishment of a full-process quality control and quality traceability system for traditional Chinese medicine. Scholars have made meaningful explorations in research models and methods. However, most research work focuses on the uniqueness and quantification of the chemical composition of raw traditional Chinese medicine products to conduct certain analysis and prediction in order to discover the quality markers of raw traditional Chinese medicine products. However, for special varieties such as processed traditional Chinese medicine products, they are quite different from raw products in terms of chemical composition and efficacy, so the quality control methods and standards of raw traditional Chinese medicine products cannot be fully applied. Therefore, how to compare the quality marker information of raw traditional Chinese medicine products with the changes after processing, find the correlation between the chemical and medicinal changes during the processing process, and obtain quality markers that can truly reflect the processing process of traditional Chinese medicine and the terminal product requires appropriate technology and methods to solve. Summary of the invention

[0004] Based on this, it is necessary to provide a method for identifying quality markers of Chinese medicine preparation to address the above issues.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for identifying quality markers of traditional Chinese medicine processing, the method comprising the following steps:

[0007] Step A: Determine the components of processed Chinese medicine that enter the blood;

[0008] Step B: Conduct network pharmacology analysis of the components entering the blood of processed Chinese medicines for targets that are closely related to the changes in pharmacodynamics before and after processing, determine the components entering the blood that are closely related to the pharmacological changes of processed Chinese medicines, and determine the primary and secondary relationships of the components;

[0009] Step C: Comparison of the components of raw Chinese medicine entering the blood with those of processed Chinese medicine entering the blood;

[0010] Step D: Identify the quality markers of processed Chinese medicines.

[0011] Furthermore, the experimental animals in step A are divided into a blank group and an experimental group, and the dosage, blood collection time and blood components are used as the evaluation indicators to conduct tests and analyses to determine the optimal blood collection time and dosing regimen;

[0012] After fasting for 12 hours, the experimental animals were randomly divided into a blank group and an experimental group. The experimental group was given medication according to the determined dosing regimen, and blood was collected at the optimal time and processed with appropriate methods. The blood components and serum fingerprints of the Chinese medicine preparations were obtained by liquid spectrometry and mass spectrometry analysis.

[0013] Furthermore, in step C, the dosage of the processed Chinese medicine is equivalent to that of the raw product, and blood is collected at the optimal blood collection time to obtain the blood component spectrum of the raw Chinese medicine, which is compared with the blood component spectrum of the processed Chinese medicine, and combined with the network pharmacology analysis of the blood components of the processed Chinese medicine, the main blood components that have undergone major changes before and after processing are determined.

[0014] Furthermore, the in vitro chemical components of the Chinese medicine preparation in step D are analyzed by liquid chromatography-mass spectrometry, and the in vitro prototype components of the main blood-entering components determined in step C are correlated according to the cleavage rules, and their prototype components, i.e., the quality markers of Chinese medicine preparation, are determined.

[0015] Compared with the prior art, the identification method of the quality marker of Chinese medicine processing of the present invention, "processing quality marker" has the following characteristics in addition to the five elements of quality marker of Chinese medicine, namely, mass transfer and traceability, component specificity, component effectiveness, component measurability and compound compatibility environment: processing quality marker is a component that is newly generated, changes in content, disappears or transforms during the processing and processing of Chinese medicine according to clinical needs under the guidance of Chinese medicine theory; processing quality marker may come from Chinese medicine raw materials or added processing auxiliary materials; processing quality marker is closely related to the change of efficacy before and after Chinese medicine processing; processing quality marker can be used for screening of Chinese medicine processing technology, processing quality evaluation and quality control. The proposal of the research method of "processing quality marker" of Chinese medicine is a research exploration of the research on the quality of Chinese medicine processing under the guidance of Chinese medicine theory, taking into account the particularity of Chinese medicine processing and the characteristics of clinical medication, and is a meaningful attempt to further explore the quality control research of Chinese medicine processing.

[0016] At present, ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) can quickly analyze the entire composition of traditional Chinese medicine and its absorption and metabolism components in the body, and provide modern analytical means and methods for further correlation analysis of in vivo and in vitro substance groups. Network pharmacology is a new method and strategy for drug design proposed on the basis of the rapid development of systems biology and multidirectional pharmacology. It involves systems biology, network biology analysis, gene connectivity and gene pleiotropy. Through network pharmacology analysis, the connection between chemical substances, pharmacological effects and targets can be established, the targets of each chemical substance can be quickly determined, and their contribution can be predicted. The development of the above modern technologies and methods has made it possible to study the "processing quality markers" of traditional Chinese medicine. DETAILED DESCRIPTION

[0017] The present invention provides a method for identifying a quality marker of traditional Chinese medicine processing, comprising the following steps:

[0018] Step A: Determine the components of processed Chinese medicine that enter the blood;

[0019] Step B: Conduct network pharmacology analysis of the components entering the blood of processed Chinese medicines for targets that are closely related to the changes in pharmacodynamics before and after processing, determine the components entering the blood that are closely related to the pharmacological changes of processed Chinese medicines, and determine the primary and secondary relationships of the components;

[0020] Step C: Comparison of the components of raw Chinese medicine entering the blood with those of processed Chinese medicine entering the blood;

[0021] Step D: Identify the quality markers of processed Chinese medicines.

[0022] Furthermore, the experimental animals in step A are divided into a blank group and an experimental group, and the dosage, blood collection time and blood components are used as the evaluation indicators to conduct tests and analyses to determine the optimal blood collection time and dosing regimen;

[0023] After fasting for 12 hours, the experimental animals were randomly divided into a blank group and an experimental group. The experimental group was given medication according to the determined dosing regimen, and blood was collected at the optimal time and processed with appropriate methods. The blood components and serum fingerprints of the Chinese medicine preparations were obtained by liquid spectrometry and mass spectrometry analysis.

[0024] Furthermore, in step C, the dosage of the processed Chinese medicine is equivalent to that of the raw product, and blood is collected at the optimal blood collection time to obtain the blood component spectrum of the raw Chinese medicine, which is compared with the blood component spectrum of the processed Chinese medicine, and combined with the network pharmacology analysis of the blood components of the processed Chinese medicine, the main blood components that have undergone major changes before and after processing are determined.

[0025] Furthermore, the in vitro chemical components of the Chinese medicine preparation in step D are analyzed by liquid chromatography-mass spectrometry, and the in vitro prototype components of the main blood-entering components determined in step C are correlated according to the cleavage rules, and their prototype components, i.e., the quality markers of Chinese medicine preparation, are determined.

[0026] For example, in one embodiment, the function of moistening the lungs, relieving cough and relieving asthma of Chinese medicine a is enhanced after processing, and the method for identifying the processing quality marker comprises the following steps:

[0027] First, determine the optimal dosage and optimal blood collection time

[0028] Rats were gavage-administered with the processed extract of Chinese medicine a at dosages of 5, 10, 20, and 30 times the conventional human dosage, and the optimal dosage was determined to be 30 times the human dosage based on the number of peaks in the chromatogram of the drug-containing serum. Blood was collected at 60, 90, 120, and 180 minutes after administration, and the optimal administration time was determined to be 120 minutes based on the number of peaks in the chromatogram of the drug-containing serum.

[0029] Secondly, obtain the blood component spectrum of processed Chinese medicine a

[0030] The drug-containing serum sample of the processed product of Chinese medicine a was put into the liquid chromatography tandem mass spectrometer for analysis. The peak interval was used as the evaluation index to reasonably select the chromatographic column, detector, chromatographic mobile phase and flow rate; the nitrogen flow rate of the mass spectrometer, CDL temperature, detector voltage, selection of internal standard and other conditions. By detecting the mass spectrum of the chromatographic peak, the ion information of the chemical components in the plasma was analyzed in general. The chromatogram of the processed serum of Chinese medicine a was obtained by comparing the retention time (tR) and analyzing the mass-to-charge ratio (m / z) of the ion fragments, and the blood components were analyzed.

[0031] Again, network pharmacology study on the blood - entering components of the processed product of traditional Chinese medicine a

[0032] (1) Prediction and collation of compound targets

[0033] Through the compound information retrieval platform, collate the basic information of the compounds obtained from the blood - entering analysis of the processed product of traditional Chinese medicine a, including the molecular formula, CAS number, structural formula, etc. of the compounds. Search for the SMILES chemical formula corresponding to the chemical structure, and use the SEAsearch server, Swiss Target Prediction, and Drugbank databases to retrieve the corresponding target information. Remove duplicates and remove the target entries of non - human species through the target name.

[0034] (2) Prediction and collation of "moistening the lungs, relieving cough and asthma" targets

[0035] Use the keywords "cough", "chronic cough", "asthma", "bronchial asthma" as search terms to search in multiple human gene databases, and export the results of these databases to an Excel spreadsheet for collation.

[0036] (3) Construction of the ingredient - disease target network

[0037] Use the Cytoscape 3.7.2 visualization software to construct the "ingredient - disease" network, and analyze the network topology - related parameters through NetworkAnalyzer.

[0038] (4) Construction of the PPI protein - protein interaction network

[0039] Import the blood - entering components of the processed product of traditional Chinese medicine a and the "moistening the lungs, relieving cough and asthma" disease - related targets into the STRING platform, select "homo sapiens", draw the protein - protein interaction network relationship (PPI) diagram, and obtain the parameters of the interaction between proteins. Then import the above - obtained data into the visualization processing software Cytoscape 3.7.2, and use the "NetworkAnalyzer" button to analyze this PPI network.

[0040] (5) KEGG pathway enrichment analysis

[0041] Import the blood - entering components of the processed product of traditional Chinese medicine a and the "cough - relieving and asthma - calming" disease - related targets into the Metascape platform, limit the species to Homo sapiens, conduct KEGG pathway enrichment analysis, and screen the top 25 pathways related to "moistening the lungs, relieving cough and asthma" to draw a bubble chart.

[0042] (6) Construction of the "drug - target - pathway action" network

[0043] The “drug-target pathway action” network was constructed using Cytoscape 3.7.2 visualization software, and the network topology-related parameters were analyzed using Network Analyzer.

[0044] (7) Molecular docking

[0045] The workflow of molecular docking virtual screening was completed in Schrödinger 2015 software. First, the MMFFs force field was used to parameterize the small molecule ligands under pH = 7.0 ± 2.0. The crystal structure of the protein complex was exported from the PDB database (https: / / www.rcsb.org / ) and preprocessed by hydrogenation, side chain completion, and structure minimization. The binding pocket was defined by the ligand binding site in the crystal, and the standard precision (SP) scoring function was used for molecular docking analysis, and the ligand in the crystal was scored for molecular docking test.

[0046] Then, the blood components of raw Chinese medicine a were compared with those of processed Chinese medicine a.

[0047] The dose of the processed Chinese medicine a was equivalent to that of the raw Chinese medicine a, and blood was collected at the optimal blood collection time to obtain the blood component spectrum of the raw Chinese medicine a. The spectrum was compared with the blood component spectrum of the processed Chinese medicine a, and combined with the network pharmacology analysis of the blood components of the processed Chinese medicine a, the blood components of the processed Chinese medicine a that had undergone major changes were found.

[0048] Finally, identify the "processing quality markers" of processed Chinese medicine a

[0049] The in vitro chemical components of the processed Chinese medicine a are analyzed by liquid spectrometry, and the in vitro prototype components of the main blood-entering components determined in the above "3" are correlated and analyzed based on the cleavage rules, and their prototype components, i.e., the "processing quality markers" of the processed Chinese medicine a, are determined.

[0050] Compared with the prior art, the identification method of the quality marker of Chinese medicine processing of the present invention, "processing quality marker" has the following characteristics in addition to the five elements of quality marker of Chinese medicine, namely, mass transfer and traceability, component specificity, component effectiveness, component measurability and compound compatibility environment: processing quality marker is a component that is newly generated, changes in content, disappears or transforms during the processing and processing of Chinese medicine according to clinical needs under the guidance of Chinese medicine theory; processing quality marker may come from Chinese medicine raw materials or added processing auxiliary materials; processing quality marker is closely related to the change of efficacy before and after Chinese medicine processing; processing quality marker can be used for screening of Chinese medicine processing technology, processing quality evaluation and quality control. The proposal of the research method of "processing quality marker" of Chinese medicine is a research exploration of the research on the quality of Chinese medicine processing under the guidance of Chinese medicine theory, taking into account the particularity of Chinese medicine processing and the characteristics of clinical medication, and is a meaningful attempt to further explore the quality control research of Chinese medicine processing.

[0051] At present, ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) can quickly analyze the entire composition of traditional Chinese medicine and its absorption and metabolism components in the body, and provide modern analytical means and methods for further correlation analysis of in vivo and in vitro substance groups. Network pharmacology is a new method and strategy for drug design proposed on the basis of the rapid development of systems biology and multidirectional pharmacology. It involves systems biology, network biology analysis, gene connectivity and gene pleiotropy. Through network pharmacology analysis, the connection between chemical substances, pharmacological effects and targets can be established, the targets of each chemical substance can be quickly determined, and their contribution can be predicted. The development of the above modern technologies and methods has made it possible to study the "processing quality markers" of traditional Chinese medicine.

[0052] The above only describes the specific embodiments of the present invention, but cannot be regarded as the protection scope of the present invention. Any equivalent changes or modifications made according to the design spirit of the present invention should be considered to fall within the protection scope of the present invention.

Claims

1. A method for identifying quality markers of traditional Chinese medicine processing, Features: The method comprises the following steps: Step A: Determine the components of processed Chinese medicines that enter the blood: determine the optimal dosage and the optimal blood collection time, and use liquid chromatography-mass spectrometry to obtain the spectrum of components of processed Chinese medicines that enter the blood; Step B: Conduct network pharmacology analysis of the components entering the blood of processed Chinese medicines for targets that are closely related to the changes in pharmacodynamics before and after processing, determine the components entering the blood that are closely related to the pharmacological changes of processed Chinese medicines, and determine the primary and secondary relationships of the components; Includes: (1) prediction and organization of compound targets; (2) Prediction and organization of disease-related targets; (3) Construction of component and disease target network; (4) Construction of PPI protein interaction network: PPI diagram of protein-protein interaction network relationship is drawn by comparing the blood components of processed Chinese medicine products with disease-related targets, the parameters of protein-protein interaction are obtained, and the PPI network is analyzed; (5) KEGG pathway enrichment analysis: KEGG pathway enrichment analysis was performed on the components of TCM processed products entering the blood and disease-related targets, and the top 25 disease-related pathways were screened and bubble charts were drawn; (6) Construction of the "drug target pathway action" network; (7) Molecular docking: parameterize the small molecule ligand, derive the crystal structure of the protein complex from the database, perform pre-processing such as hydrogenation, side chain completion, and structure minimization, define the binding pocket based on the ligand binding site in the crystal, perform molecular docking analysis, and perform molecular docking test and scoring on the ligand in the crystal; Step C: Comparison of the components of raw Chinese medicine entering the blood with those of processed Chinese medicine entering the blood: The dose of processed Chinese medicine is equivalent to that of raw Chinese medicine, and blood is collected at the optimal blood collection time to obtain the spectrum of blood components of raw Chinese medicine, and then compared with the spectrum of blood components of processed Chinese medicine. Combined with the network pharmacology analysis of the blood components of processed Chinese medicine, the blood components that have undergone major changes in processed Chinese medicine are found; Step D: Identify the quality markers of processed Chinese medicines: The in vitro chemical components of processed Chinese medicines are analyzed by liquid spectrometry, and the in vitro prototype components of the main blood-entering components are correlated and analyzed based on the cleavage rules, and their prototype components, namely the "processing quality markers" of processed Chinese medicines, are determined.

2. A method for identifying a quality marker of Chinese medicine processing according to claim 1, Features: In step A, the experimental animals are divided into a blank group and an experimental group, and the dosage, blood collection time and blood components are used as the evaluation indicators to conduct tests and analyses to determine the optimal blood collection time and dosing regimen; After fasting for 12 hours, the experimental animals were randomly divided into a blank group and an experimental group. The experimental group was given medication according to the determined dosing regimen, and blood was collected at the optimal time and processed with appropriate methods. The blood components and serum fingerprints of the Chinese medicine preparations were obtained by liquid spectrometry and mass spectrometry analysis.

3. A method for identifying a quality marker of Chinese medicine processing according to claim 2, Features: In the step C, the dosage of the processed Chinese medicine is equivalent to that of the raw product, and blood is collected at the optimal blood collection time to obtain the blood component spectrum of the raw Chinese medicine, which is compared with the blood component spectrum of the processed Chinese medicine. Combined with the network pharmacology analysis of the blood components of the processed Chinese medicine, the main blood components that have undergone major changes before and after the processing are determined.

4. A method for identifying a quality marker of Chinese medicine processing according to claim 3, Features: In the step D, the in vitro chemical components of the Chinese medicine processed product are analyzed by liquid chromatography-mass spectrometry, and the in vitro prototype components of the main blood-entering components determined in step C are correlated and analyzed according to the cleavage rules, and their prototype components, i.e., the quality markers of Chinese medicine processing, are determined.