A method for studying the effective components of Liuwei Buqi prescription and its application

Abstract

The present invention provides a method for studying the effective components of Liuwei Buqi Fang. The traditional Chinese medicine formula of Liuwei Buqi Fang is: ginseng, astragalus, alpinia oxyphylla, polygonatum odoratum, tangerine peel and cinnamon bark. The research method of the present invention highlights clinical application, takes revealing the biological essence corresponding to the Liuwei Buqi Fang syndrome as a link, actively explores the core molecular mechanism of Liuwei Buqi Fang in treating COPD, and has clear research objectives and innovative research ideas. The scheme design takes the prevention and treatment of COPD as the primary priority, reflects the intervention and management of COPD based on stages and syndromes, highlights clinical application, and reflects the original thinking of traditional Chinese medicine in syndrome differentiation and treatment and disease-syndrome combination. UPLC-Q / TOF-MS technology was used to characterize the Liuwei Buqi Fang extract and blood-entering components, and a panoramic depiction of the effective component group of Xin'an Liuwei Buqi Fang was obtained. In combination with the pharmacodynamics research of COPD and urine metabolomics, its mechanism of action was explained, and the material basis of the efficacy of traditional Chinese medicine was revealed from the level of component-drug efficacy combination.

Description

Technical Field

[0001] The invention relates to the technical field of research on components of traditional Chinese medicines, and in particular to a method for researching effective components of a six-flavor qi-replenishing prescription and its application. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a major disease with high morbidity, disability and mortality rates worldwide. According to the "China Chronic Obstructive Pulmonary Disease Grading Diagnosis and Treatment Report (2020)", there are nearly 100 million COPD patients in China, and the high-risk population for COPD in the population aged 40 and above accounts for 20.51%, and the prevalence of COPD in high-risk groups has reached 33.4%. The prevalence of COPD is showing a significant upward trend, and the prevention and control situation is severe.

[0003] Lung Qi deficiency is the fundamental factor in the occurrence and development of lung diseases such as COPD. If the disease is prolonged and the Qi is damaged for a long time, the lung and kidney Qi will be deficient, resulting in the lung losing its regulation and the kidney losing its sealing and storage. The blood does not circulate normally and becomes stasis. Abnormal metabolism of body fluids leads to phlegm and fluid. Blood stasis, phlegm turbidity and fluid are intertwined. Guided by the idea of ​​strengthening the foundation and nourishing the essence, the key to strengthening the foundation lies in the lungs, spleen and kidneys, and the key to nourishing the essence lies in replenishing Qi, mainly replenishing the Qi of the three organs of the lungs, spleen and kidneys. The Liuwei Buqi Recipe developed under the guidance of the idea of ​​strengthening the foundation and nourishing the essence has achieved certain therapeutic effects in the treatment of lung diseases such as COPD.

[0004] The study of the effectiveness of traditional Chinese medicine is guided by the dialectical thinking of traditional Chinese medicine. It usually uses phytochemical methods to extract the effective part compound group or monomer compound, and then confirms it through pharmacological experiments, and finally transitions to clinical trials. Due to the complexity and diversity of the ingredients in traditional Chinese medicine formulas and the complicated material basis of the efficacy, the mechanism of action of traditional Chinese medicine is unclear. Therefore, it is of great clinical significance to comprehensively study the therapeutic effects of the effective components in Liuwei Buqi Fang on related diseases; and clarifying the material basis of its key pharmacodynamic effects is particularly important for in-depth analysis of the target pathway and other mechanism studies of its disease treatment, providing technical support for the secondary development of traditional Chinese medicine and the research and development of innovative drugs, and promoting the modernization of traditional Chinese medicine. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a method for studying the effective components of Liuwei Buqi Fang, revealing the correlation between the pharmacodynamic material basis, activity and efficacy of Liuwei Buqi Fang in treating COPD, so as to solve the problems raised in the above background technology.

[0006] The technical problem solved by the present invention is achieved by the following technical scheme: a method for studying the effective components of Liuwei Buqi prescription, the Chinese medicine formula of Liuwei Buqi prescription is: ginseng, astragalus, Alpinia oxyphylla, polygonatum odoratum, tangerine peel and cinnamon bark, and the research includes the following aspects:

[0007] Research on the material basis of the efficacy of Liuwei Buqi prescription:

[0008] (1) Analysis of the in vitro chemical components of Liuwei Buqi Fang based on UPLC-QE-Orbitrap-MS technology;

[0009] (2) Analysis of blood-transmitted components of Liuwei Buqi Recipe based on UPLC-Q-TOF-MS technology;

[0010] (3) Screening of pharmacological substances of Liuwei Buqi Formula based on network target analysis and molecular docking technology;

[0011] Research on new drug components of Liuwei Buqi Fang:

[0012] (1) Reproduce the COPD evolution model of the lung organoid microphysiological system;

[0013] (2) Innovate a systematic evaluation system for COPD organoid Chinese medicine compound prescriptions and clarify the components of new drugs;

[0014] Quality assessment of the effective components of Liuwei Buqi Fang:

[0015] (1) Identify and separate the active ingredients and doping components of traditional Chinese medicines by surface enhanced Raman spectroscopy (SERS);

[0016] (2) The peak spectrum attribution of traditional Chinese medicine was determined by surface enhanced Raman spectroscopy (SERS) technology.

[0017] As a further solution of the present invention: in vitro chemical component analysis of Liuwei Buqi Recipe based on UPLC-QE-Orbitrap-MS technology, the Liuwei Buqi Recipe drugs are finely ground, accurately weighed, and pre-treated by dissolution, ultrasonic extraction, centrifugation, and supernatant collection to prepare test solution and corresponding reference solution;

[0018] Ultra performance liquid chromatography-quadrupole-orbitrap tandem high resolution mass spectrometry (UPLC-QE-Orbitrap-MS) technology was used to characterize the chemical components of the two prescriptions; the raw data in RAW format were processed by Compound Discoverer software, and the accurate mass, elemental composition, isotope spectrum and MS2 spectrum of the mzCloud database and TCM library database were used to identify each chromatographic peak; the fragment ion information obtained by Xcalibur software was compared with the MS2 and MASSBANK database of the reference substance to further infer and confirm the compounds, summarize the compound fragmentation rules, and analyze the chemical components of the in vitro effective substances of Liuwei Buqi Recipe, laying a foundation for screening its in vivo components.

[0019] As a further scheme of the present invention: analysis of the components migrating in the blood of Liuwei Buqi Recipe based on UPLC-Q-TOF-MS technology, high-throughput rapid analysis of the chemical components of Liuwei Buqi Recipe based on UPLC-QE-Orbitrap-MS technology, combined with ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology, analysis of the retention time, mass-to-nuclear ratio, fragment ions and other information of the components entering the blood of Liuwei Buqi Recipe, comparison, calculation and inference of the differential substances in the recipe with the information in the database or literature, obtaining detailed information on the prototype components and the components entering the blood, thereby further inferring the potential metabolic pathways and targets of Liuwei Buqi Recipe in the body after oral administration.

[0020] As a further scheme of the present invention: screening of pharmacological substances of Liuwei Buqi Recipe based on network target analysis and molecular docking technology, based on the components migrating in the blood, component-disease target correlation analysis was performed on the components entering the blood of Liuwei Buqi Recipe and COPD disease, and by constructing a "core target-component" network, the material basis of the prescription for treating COPD was predicted, the predicted active ingredients were molecularly docked with the targets for treating COPD, and the components with a high degree of match were selected as potential pharmacological substances.

[0021] UPLC-Q / TOF-MS technology was used to characterize the extract and blood components of Liuwei Buqi Fang, and a comprehensive picture of the effective ingredient group of Liuwei Buqi Fang was depicted. Combined with the pharmacodynamics research of COPD and urine metabolomics, its mechanism of action was explained, revealing the material basis of the efficacy of traditional Chinese medicine from the level of component-efficacy combination.

[0022] As a further solution of the present invention: reproducing the COPD evolution model of the lung organoid microphysiological system, comprising the following steps:

[0023] (1) Microfluidic chip fabrication method: Microfluidic chip devices are all fabricated using classical soft lithography methods. The fabrication process mainly includes mask design, template fabrication, and chip processing.

[0024] (2) Preparation of PDMS gasket: prepare PDMS working solution, dry, cut into strips, drip working solution, bake, cut, punch, soak in 75% ethanol solution, evacuate, dry and set aside.

[0025] (3) Construction of 3D lung organoid model: The primary cell suspension from normal lung tissue was added to the matrix gel, then inoculated with the matrix gel mixture, allowed to solidify, and the complete organoid culture medium was slowly added. When the organoids were larger than 50 μ and no longer increased in size, the organoids were collected on a microfluidic chip for subsequent experiments.

[0026] (4) Construction of COPD organoid platform: Based on the 3D model of lung organoids, modern technology is used to construct a COPD organoid model of lung and kidney qi deficiency syndrome.

[0027] As a further solution of the present invention: Innovating a systematic evaluation system for COPD organoid Chinese medicine compound to clarify the new drug components includes the following steps:

[0028] (1) UPLC-QTOF / MS was used to screen the active ingredients of Liuwei Buqi Recipe that enter the blood, and its efficacy was determined based on the COPD lung organoid level: UPLC-QTOF / MS results were used to screen the active monomer components with high blood content and stable properties, and the candidate components were tested for efficacy using the COPD lung organoid model. The components with obvious efficacy were retained, and the components with insignificant effects / no efficacy were eliminated, thus locking in the active ingredients that enter the blood.

[0029] (2) Optimize the combination arrangement of active ingredients of traditional Chinese medicine and clarify their "efficacy": Use network pharmacology, bioinformatics and orthogonal design methods to combine the active ingredients, and perform pharmacodynamic testing using the COPD lung organoid model to screen for the optimal active ingredient group with a "synergistic" anti-COPD effect.

[0030] (3) Systematically reveal the pharmacodynamic material basis, activity, and efficacy correlation of traditional Chinese medicine compound prescriptions for the treatment of COPD: through small molecule binding experiments, clarify the protein targets of the relevant active pharmacological ingredients; and use structural biology methods to analyze the crystal structure of the pharmacological ingredient-protein complex, while combining relevant mutants to explore the effects of active ingredients on target functions.

[0031] As a further solution of the present invention: surface enhanced Raman spectroscopy (SERS) technology is used to identify and separate the effective ingredients and doped components of traditional Chinese medicine, and the chemical components of Liuwei Buqi Fang are separated respectively using the preferred silver sol as the enhanced substrate and TCL as the detection method, and the separated trace elements are identified using SERS technology, and the doped chemical components in Liuwei Buqi Fang are detected by TCL-SERS technology.

[0032] As a further solution of the present invention: surface enhanced Raman spectroscopy (SERS) technology is used to determine the peak spectrum attribution of traditional Chinese medicine. Samples of Liuwei Buqi Recipe are detected using gold film, silver nanoparticles, and gold film-silver nanoparticle enhanced substrates, respectively. The Raman signal of the drug is observed using SERS technology to obtain the preliminary peak spectrum attribution of the sample.

[0033] As a further solution of the present invention: the Liuwei Buqi recipe also includes a preliminary study of components, including the following steps:

[0034] (1) Study on the rationality of the formula: Conduct literature analysis on Liuwei Buqi Fang, systematically summarize and analyze its TCM clinical data, chemical components, extraction and separation methods, pharmacological effects, etc., and determine the rationality of its formula from the perspective of literature analysis; study the changes in the content of the main chemical components in the Liuwei Buqi Fang compatibility process, and study the rationality of the Liuwei Buqi Fang formula from the perspective of changes in the compatibility index components;

[0035] (2) Preparation process research: The water absorption rate of Chinese herbal medicine slices was investigated, and the water extraction process was optimized by comprehensive scoring method; the dry paste yield and the content of a certain index component of Liuwei Buqi Recipe were used as evaluation indicators to compare the impurity removal effects of different water extraction methods, select the best refining process, and screen the most effective drying method based on this; through the investigation of new excipients, the particle formability, moisture absorption rate, bulk density and repose angle were used as indicators to select the best excipients and appropriate excipient dosage, and screen the most effective granulation method;

[0036] (3) Quality standard research: Thin layer identification research was conducted on several medicinal materials in Liuwei Buqi Fang, and the corresponding negative control solution without flavor was prepared at the same time to prove the specificity of the method; according to the relevant provisions of the capsule item of the "Chinese Pharmacopoeia", the water content, filling volume difference, disintegration time, heavy metals, Kun salt and microbial limit test method of Liuwei Buqi Fang were checked; with reference to the "Chinese Pharmacopoeia" and relevant literature, HPLC method was used to quantify multiple components simultaneously and the fingerprint spectrum of Liuwei Buqi Fang was studied;

[0037] (4) Preliminary stability test: According to the relevant provisions of the "Technical Requirements for Quality Stability Research of New Chinese Medicines" in the "Technical Requirements for Research of New Chinese Medicines" issued by the State Food and Drug Administration, a stability test was conducted on the Liuwei Buqi Recipe, with key inspection items including the appearance of the product, color, content, related substances, disintegration time or solubility, and water content;

[0038] (5) Homogeneity test: The HPLC method was used to measure and analyze the uniformity of the quality of Liuwei Buqi Fang between different batches.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows: the Chinese medicinal ingredients of the Liuwei Buqi Recipe are ginseng, astragalus, alpinia oxyphylla, polygonatum odoratum, tangerine peel and cinnamon bark, and it is mainly used to treat COPD lung and kidney qi deficiency syndrome. Clinical observations show that it can delay the decline of patients' lung function to varying degrees, enhance immunity, reduce lung inflammatory response, and reduce the number of acute exacerbations.

[0040] The research method of the present invention highlights clinical application, takes revealing the biological essence corresponding to Liuwei Buqi Fang as a link, actively explores the core molecular mechanism of Liuwei Buqi Fang in treating COPD, has clear research objectives, and innovative research ideas. The design of the program takes the prevention and treatment of COPD as the primary priority, reflects the intervention and management of COPD by stages and syndromes, highlights clinical application, and reflects the original thinking of traditional Chinese medicine in syndrome differentiation and treatment, and disease and syndrome combination. UPLC-Q / TOF-MS technology was used to characterize the Liuwei Buqi Fang extract and blood components, and a panoramic depiction of the effective ingredient group of Liuwei Buqi Fang was obtained. In combination with the pharmacodynamics study of COPD and urine metabolomics, its mechanism of action was explained, and the material basis of the efficacy of traditional Chinese medicine was revealed from the level of component-effect combination. The new drug component research reproduced the COPD evolution model of lung organoids, and the pharmacodynamics test was carried out through the COPD lung organoid model, and then the optimal effective ingredient group with anti-COPD effect was screened, revealing the pharmacodynamic material basis-activity-efficacy correlation of traditional Chinese medicine compound in treating COPD; surface-enhanced Raman spectroscopy technology was used to evaluate the overall effective components. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the technical roadmap of the research content of this invention. DETAILED DESCRIPTION

[0042] In order to make the technical means for realizing the present invention, the creative features, the objectives and effects to be achieved easily understood, the present invention is further described below with reference to specific diagrams.

[0043] like Figure 1 As shown, this embodiment provides a method for studying the effective components of Liuwei Buqi Recipe, the Chinese medicine composition of Liuwei Buqi Recipe is: ginseng, astragalus, Alpinia oxyphylla, polygonatum odoratum, tangerine peel and cinnamon bark, and the research includes the following aspects:

[0044] Research on the material basis of the efficacy of Liuwei Buqi prescription:

[0045] (1) Analysis of the in vitro chemical components of Liuwei Buqi Fang based on UPLC-QE-Orbitrap-MS technology;

[0046] (2) Analysis of blood-transmitted components of Liuwei Buqi Recipe based on UPLC-Q-TOF-MS technology;

[0047] (3) Screening of pharmacological substances of Liuwei Buqi Formula based on network target analysis and molecular docking technology;

[0048] Research on new drug components of Liuwei Buqi Fang:

[0049] (1) Reproduce the COPD evolution model of the lung organoid microphysiological system;

[0050] (2) Innovate a systematic evaluation system for COPD organoid Chinese medicine compound prescriptions and clarify the components of new drugs;

[0051] Quality assessment of the effective components of Liuwei Buqi Fang:

[0052] (1) Identify and separate the active ingredients and doping components of traditional Chinese medicines by surface enhanced Raman spectroscopy (SERS);

[0053] (2) The peak spectrum attribution of traditional Chinese medicine was determined by surface enhanced Raman spectroscopy (SERS) technology.

[0054] In vitro chemical composition analysis of Liuwei Buqi Recipe based on UPLC-QE-Orbitrap-MS technology: The Liuwei Buqi Recipe drugs were finely ground, accurately weighed, and pre-treated by dissolution, ultrasonic extraction, centrifugation, and supernatant collection to prepare test solution and corresponding reference solution;

[0055] Ultra performance liquid chromatography-quadrupole-orbitrap tandem high resolution mass spectrometry (UPLC-QE-Orbitrap-MS) technology was used to characterize the chemical components of the two prescriptions; the raw data in RAW format were processed by Compound Discoverer software, and the accurate mass, elemental composition, isotope spectrum and MS2 spectrum of the mzCloud database and TCM library database were used to identify each chromatographic peak; the fragment ion information obtained by Xcalibur software was compared with the MS2 and MASSBANK database of the reference substance to further infer and confirm the compounds, summarize the compound fragmentation rules, and analyze the chemical components of the in vitro effective substances of Liuwei Buqi Recipe, laying a foundation for screening its in vivo components.

[0056] Analysis of the components migrating into the blood of Liuwei Buqi Fang based on UPLC-Q-TOF-MS technology, high-throughput rapid analysis of the chemical components of Liuwei Buqi Fang based on UPLC-QE-Orbitrap-MS technology, combined with ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology, analysis of the retention time, mass-to-nuclear ratio, fragment ions and other information of the components entering the blood of Liuwei Buqi Fang, comparison, calculation and inference of the differential substances in the prescription with the information in the database or literature, and obtaining detailed information on the prototype components and the components entering the blood, so as to further infer the potential metabolic pathways and targets of Liuwei Buqi Fang in the body after oral administration.

[0057] The pharmacological substances screening of Liuwei Buqi Decoction was carried out based on network target analysis and molecular docking technology. Based on the components migrating in the blood, the component-disease target correlation analysis of the Liuwei Buqi Decoction entering the blood and COPD disease was carried out. By constructing a "core target-component" network, the material basis of the prescription for treating COPD was predicted, and the predicted active ingredients were molecularly docked with the targets for treating COPD, and the components with high matching degree were selected as potential pharmacological substances.

[0058] UPLC-Q / TOF-MS technology was used to characterize the extract and blood components of Liuwei Buqi Fang, and a comprehensive picture of the effective ingredient group of Liuwei Buqi Fang was depicted. Combined with the pharmacodynamics research of COPD and urine metabolomics, its mechanism of action was explained, revealing the material basis of the efficacy of traditional Chinese medicine from the level of component-efficacy combination.

[0059] Reproducing the COPD evolution model of the lung organoid microphysiological system includes the following steps:

[0060] (1) Microfluidic chip fabrication method: Microfluidic chip devices are all fabricated using classical soft lithography methods. The fabrication process mainly includes mask design, template fabrication, and chip processing.

[0061] (2) Preparation of PDMS gasket: prepare PDMS working solution, dry, cut into strips, drip working solution, bake, cut, punch, soak in 75% ethanol solution, evacuate, dry and set aside.

[0062] (3) Construction of 3D lung organoid model: The primary cell suspension from normal lung tissue was added to the matrix gel, then inoculated with the matrix gel mixture, allowed to solidify, and the complete organoid culture medium was slowly added. When the organoids were larger than 50 μ and no longer increased in size, the organoids were collected on a microfluidic chip for subsequent experiments.

[0063] (4) Construction of COPD organoid platform: Based on the 3D model of lung organoids, modern technology is used to construct a COPD organoid model of lung and kidney qi deficiency syndrome.

[0064] Innovate the systematic evaluation system of COPD organoid Chinese medicine compound and clarify the components of new drugs, including the following steps:

[0065] (1) UPLC-QTOF / MS was used to screen the active ingredients of Liuwei Buqi Recipe that enter the blood, and its efficacy was determined based on the COPD lung organoid level: UPLC-QTOF / MS results were used to screen the active monomer components with high blood content and stable properties, and the candidate components were tested for efficacy using the COPD lung organoid model. The components with obvious efficacy were retained, and the components with insignificant effects / no efficacy were eliminated, thus locking in the active ingredients that enter the blood.

[0066] (2) Optimize the combination arrangement of active ingredients of traditional Chinese medicine and clarify their "efficacy": Use network pharmacology, bioinformatics and orthogonal design methods to combine the active ingredients, and perform pharmacodynamic testing using the COPD lung organoid model to screen for the optimal active ingredient group with a "synergistic" anti-COPD effect.

[0067] (3) Systematically reveal the pharmacodynamic material basis, activity, and efficacy correlation of traditional Chinese medicine compound prescriptions for the treatment of COPD: through small molecule binding experiments, clarify the protein targets of the relevant active pharmacological ingredients; and use structural biology methods to analyze the crystal structure of the pharmacological ingredient-protein complex, while combining relevant mutants to explore the effects of active ingredients on target functions.

[0068] Surface enhanced Raman spectroscopy (SERS) technology was used to identify and separate the effective ingredients and doped components of traditional Chinese medicine. The chemical components of Liuwei Buqi Fang were separated using the preferred silver sol as the enhancement substrate and TCL as the detection method. The SERS technology was used to identify the separated trace elements, and the doped chemical components in Liuwei Buqi Fang were detected using the TCL-SERS technology.

[0069] Surface enhanced Raman spectroscopy (SERS) technology was used to determine the peak spectrum attribution of traditional Chinese medicine. Samples of Liuwei Buqi Recipe were detected using gold film, silver nanoparticles, and gold film-silver nanoparticle enhanced substrates, respectively. The SERS technology was used to observe the Raman signals of the drugs and obtain the preliminary peak spectrum attribution of the samples.

[0070] In this embodiment, the Liuwei Buqi recipe also includes a preliminary study of components, including the following steps:

[0071] (1) Study on the rationality of the formula: Conduct literature analysis on Liuwei Buqi Fang, systematically summarize and analyze its TCM clinical data, chemical components, extraction and separation methods, pharmacological effects, etc., and determine the rationality of its formula from the perspective of literature analysis; study the changes in the content of the main chemical components in the Liuwei Buqi Fang compatibility process, and study the rationality of the Liuwei Buqi Fang formula from the perspective of changes in the compatibility index components;

[0072] (2) Preparation process research: The water absorption rate of Chinese herbal medicine slices was investigated, and the water extraction process was optimized by comprehensive scoring method; the dry paste yield and the content of a certain index component of Liuwei Buqi Recipe were used as evaluation indicators to compare the impurity removal effects of different water extraction methods, select the best refining process, and screen the most effective drying method based on this; through the investigation of new excipients, the particle formability, moisture absorption rate, bulk density and repose angle were used as indicators to select the best excipients and appropriate excipient dosage, and screen the most effective granulation method;

[0073] (3) Quality standard research: Thin layer identification research was conducted on several medicinal materials in Liuwei Buqi Fang, and the corresponding negative control solution without flavor was prepared at the same time to prove the specificity of the method; according to the relevant provisions of the capsule item of the "Chinese Pharmacopoeia", the water content, filling volume difference, disintegration time, heavy metals, Kun salt and microbial limit test method of Liuwei Buqi Fang were checked; with reference to the "Chinese Pharmacopoeia" and relevant literature, HPLC method was used to quantify multiple components simultaneously and the fingerprint spectrum of Liuwei Buqi Fang was studied;

[0074] (4) Preliminary stability test: According to the relevant provisions of the "Technical Requirements for Quality Stability Research of New Chinese Medicines" in the "Technical Requirements for Research of New Chinese Medicines" issued by the State Food and Drug Administration, a stability test was conducted on the Liuwei Buqi Recipe, with key inspection items including the appearance of the product, color, content, related substances, disintegration time or solubility, and water content;

[0075] (5) Homogeneity test: The HPLC method was used to measure and analyze the uniformity of the quality of Liuwei Buqi Fang between different batches.

[0076] The above shows and describes the basic principle and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents. It should be noted that, in this article, if there are relational terms such as first and second, it is only used to distinguish one entity or operation from another entity or operation, and does not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the term "includes", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, the elements defined by the sentence "including one..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

Claims

1. A method for studying the effective components of Liuwei Buqi Recipe, wherein the traditional Chinese medicine composition of Liuwei Buqi Recipe is: ginseng, astragalus, alpinia oxyphylla, polygonatum odoratum, tangerine peel and cinnamon bark, characterized in that: The research includes the following aspects: Research on the material basis of the efficacy of Liuwei Buqi prescription: (1) Analysis of in vitro chemical components of Liuwei Buqi Fang based on UPLC-QE-Orbitrap-MS technology; (2) Analysis of blood-transmitted components of Liuwei Buqi Formula based on UPLC-Q-TOF-MS technology; (3) Screening of pharmacological substances of Liuwei Buqi Formula based on network target analysis and molecular docking technology; Research on new drug components of Liuwei Buqi Fang: (1) Reproduce a COPD evolution model that reproduces the microphysiological system of lung organoids; (2) Clarify the components of new drugs through a systematic evaluation system for COPD organoid Chinese medicine compound prescriptions; Quality assessment of the effective components of Liuwei Buqi Fang: (1) Identify and separate the active ingredients and doping components of traditional Chinese medicines by surface enhanced Raman spectroscopy; (2) Determine the peak spectrum of traditional Chinese medicine by surface enhanced Raman spectroscopy; In vitro chemical composition analysis of Liuwei Buqi Fang based on UPLC-QE-Orbitrap-MS technology: The Liuwei Buqi Fang drugs were finely ground, accurately weighed, dissolved, ultrasonically extracted, centrifuged, and the supernatant was taken to prepare the test solution and the corresponding reference solution; UPLC-QE-Orbitrap-MS technology was used to characterize the chemical components of Liuwei Buqi Fang. The raw data in RAW format were processed by Compound Discoverer software, and the accurate mass, elemental composition, isotope spectrum and MS2 spectrum of mzCloud database and TCM library database were identified to preliminarily identify each chromatographic peak. The fragment ion information obtained by Xcalibur software was compared with the MS2 and MASSBANK database of the reference substance to further infer and confirm the compounds, summarize the compound fragmentation rules, and analyze the chemical composition of the in vitro effective substances of Liuwei Buqi Fang, laying a foundation for screening its in vivo components. Analysis of the components of Liuwei Buqi Recipe that migrate into the blood based on UPLC-Q-TOF-MS technology, and high-throughput rapid analysis of the chemical components of Liuwei Buqi Recipe based on UPLC-QE- Orbitrap-MS technology. Combined with UPLC-Q-TOF-MS technology, the retention time, mass-to-charge ratio, and fragment ions of the components that enter the blood of Liuwei Buqi Recipe are analyzed, and the differential substances in the prescription are compared, calculated, and inferred with the information in the database or literature, and the detailed information of the prototype components and the components that enter the blood are obtained, so as to further infer the potential metabolic pathways and targets of Liuwei Buqi Recipe in the body after oral administration; The screening of the pharmacological substances of Liuwei Buqi prescription based on the network target analysis and molecular docking technology, based on the blood-transmitting components, the component-disease target correlation analysis of the Liuwei Buqi prescription blood-entering components and COPD disease was carried out. By constructing a "core target-component" network, the material basis of the prescription for treating COPD was predicted, and the predicted active components were molecularly docked with the targets for treating COPD, and the components with high matching degree were selected as potential pharmacological substances; Surface enhanced Raman spectroscopy was used to identify and separate the effective ingredients and doping components of traditional Chinese medicine. Silver sol was used as the enhancement substrate and TLC was used as the detection method to separate the chemical components of Liuwei Buqi Fang. The separated trace elements were identified using SERS technology. The doping chemical components in Liuwei Buqi Fang were detected by TLC-SERS technology. The Liuwei Buqi Recipe also includes preliminary research on components, including the following steps: (1) Study on the rationality of the formula: Conduct literature analysis on Liuwei Buqi Fang, systematically summarize and analyze its TCM clinical data, chemical components, extraction and separation methods, and pharmacological effects, and determine the rationality of its formula from the perspective of literature analysis; study the changes in the content of the main chemical components during the Liuwei Buqi Fang compatibility process, and study the rationality of the Liuwei Buqi Fang formula from the perspective of changes in the compatibility index components; (2) Preparation process research: The water absorption rate of Chinese herbal medicine slices was investigated, and the water extraction process was selected by comprehensive scoring method; the dry paste yield and the content of a certain index component of Liuwei Buqi Recipe were used as evaluation indicators to compare the impurity removal effects of different water extraction methods, select the best refining process, and screen the most effective drying method based on this; through the investigation of excipients, the particle formability, moisture absorption rate, bulk density and angle of repose were used as indicators to select the best excipients and appropriate excipient dosage, and screen the most effective granulation method; (3) Quality standard research: Thin layer identification research was conducted on several medicinal materials in Liuwei Buqi Fang, and the corresponding negative control solution without flavor was prepared at the same time to prove the specificity of the method; according to the relevant provisions of the capsule item of the "Chinese Pharmacopoeia", the moisture content, filling volume difference, disintegration time, heavy metals, Kun salt and microbial limit of Liuwei Buqi Fang were checked; with reference to the "Chinese Pharmacopoeia" and relevant literature, HPLC method was used to quantify multiple components simultaneously and the fingerprint spectrum of Liuwei Buqi Fang was studied; (4) Preliminary stability test: According to the relevant provisions of "Technical Requirements for Quality Stability Research of New Chinese Medicines" in the "Technical Requirements for Research of New Chinese Medicines" issued by the State Food and Drug Administration, the stability test of Liuwei Buqi Fang was conducted, and the inspection items included the appearance of the product, color, content, related substances, disintegration time or solubility, and water content; (5) Homogeneity test: HPLC method was used to determine the homogeneity of the quality of Liuwei Buqi Fang between different batches; Through the systematic evaluation system of COPD organoid Chinese medicine compound, the new drug components are identified, including the following steps: (1) Using UPLC-QTOF-MS to screen the active ingredients of Liuwei Buqi Recipe that enter the blood, and clarify their efficacy based on the COPD lung organoid level: Using the UPLC-QTOF-MS results, screen the active monomer ingredients with high blood content and stable properties, and then test the efficacy of the candidate ingredients using the COPD lung organoid model. The ingredients with obvious efficacy are retained, and the ingredients with insignificant effect / no efficacy are eliminated, thus locking in the active ingredients that enter the blood; (2) Optimize the combination arrangement of active ingredients of traditional Chinese medicine and clarify their "efficacy": Use network pharmacology, bioinformatics and orthogonal design methods to combine active ingredients, and conduct pharmacodynamic tests through COPD lung organoid models to screen the optimal active ingredient group with "synergistic" anti-COPD effect; (3) Systematically reveal the pharmacodynamic material basis, activity, and efficacy correlation of traditional Chinese medicine compound for COPD treatment: through small molecule binding experiments, clarify the protein targets of relevant active pharmacological ingredients; and use structural biology methods to analyze the crystal structure of the pharmacological ingredient-protein complex, and at the same time combine relevant mutants to explore the effect of active ingredients on target function.

2. A method for studying the effective components of Liuwei Buqi Recipe according to claim 1, characterized in that: Reproducing the COPD evolution model of the lung organoid microphysiological system includes the following steps: (1) Microfluidic chip fabrication method: Microfluidic chip devices are all fabricated using soft lithography, and the fabrication process includes mask design, template preparation, and chip processing; (2) Preparation of PDMS gasket: prepare PDMS working solution, dry, cut into strips, drip working solution, bake, cut, punch, soak in 75% ethanol solution, evacuate, dry and set aside; (3) Construction of 3D lung organoid model: Add the primary cell suspension from normal lung tissue into the matrix gel, then inoculate with the matrix gel mixture, solidify, and slowly add the complete organoid culture medium. When the organoids no longer grow, collect the organoids into a microfluidic chip for subsequent experiments. (4) Construction of COPD organoid platform: Based on the 3D lung organoid model, a COPD organoid model of lung and kidney qi deficiency syndrome was constructed.

3. A method for studying the effective components of Liuwei Buqi Recipe according to claim 2, characterized in that: Surface enhanced Raman spectroscopy technology was used to determine the peak spectrum attribution of traditional Chinese medicine. Samples of Liuwei Buqi Recipe were detected using gold film, silver nanoparticles, and gold film-silver nanoparticle enhanced substrates, respectively. The SERS technology was used to observe the Raman signals of the drugs to obtain the preliminary peak spectrum attribution of the samples.

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