Mass spectrum imaging method and application of traditional Chinese medicine in-blood pharmacodynamic substance of codonopsis pilosula root

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, and discloses a codonopsis pilosula root traditional Chinese medicine in-blood pharmacodynamic substance mass spectrometry imaging method and application thereof, and the method comprises the following steps: S1, pharmacodynamic research and serum drug chemical analysis of codonopsis pilosula treatment diseases: (1) establishment of an animal model: taking a spleen deficiency model as an example, (2) serum drug chemical analysis, and (3) establishment of an animal model: taking a spleen deficiency model as an example; and S2, researching the distribution of the blood-entering components in the traditional Chinese medicine. Differential distribution of various medicinal effect substances in different tissues of the plant is accurately identified and positioned by utilizing a mass spectrum imaging technology, and deep mass spectrum imaging analysis is quickly performed on the tissues of the target micro-region, so that the type and content information of the components entering the blood is obtained; according to the technology, space metabonomics is added, and the whole action mechanism of the codonopsis pilosula for treating diseases is explored, including codonopsis pilosula pharmacodynamics research and searching of pharmacodynamic substance components and spatial distribution of the pharmacodynamic substance components in codonopsis pilosula roots.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a mass spectrometry imaging method and application of blood-entering pharmacodynamic substances of codonopsis root traditional Chinese medicine. Background Art

[0002] Codonopsis pilosula is one of the most popular traditional herbal medicines in clinical practice. It has the effects of strengthening the spleen, nourishing the lungs, nourishing blood and promoting the production of body fluid. Modern medical research shows that Codonopsis pilosula can be used for spleen deficiency syndrome, coronary heart disease, gastric ulcer, regulating immune function, delaying aging and improving learning ability. The chemical composition of Codonopsis pilosula is the material basis for the formation of Codonopsis pilosula quality. Studies have shown that the chemical composition of Codonopsis pilosula is closely related to the type, climate of origin, grade and part of Codonopsis pilosula. Although Codonopsis pilosula contains a variety of chemical components such as alkaloids, alkynes, terpenes, flavonoids and sugars, there are no indicator substances for quality evaluation in the Chinese Pharmacopoeia. Therefore, the study of Codonopsis pilosula pharmacodynamic substances has begun to attract attention. On the basis of obtaining information on the chemical composition of Codonopsis pilosula, further studying the spatial distribution of chemical components in the root of Codonopsis pilosula is of great significance for understanding the migration and synthesis of chemical components in the agricultural production of Codonopsis pilosula, as well as the systematic separation of Codonopsis pilosula and the precise design of its composition and administration formula.

[0003] High spatial resolution images provide important clues for understanding the biosynthetic pathways of substances. Spatial metabolomics can fully understand the synthesis and regulatory mechanisms of pharmacological substances under growth and development and abiotic stress. Therefore, studying the spatial distribution information of pharmacological substances that enter the blood in Codonopsis root can visualize the active ingredients that play a role, which is closely related to the extraction and separation of Codonopsis active substances, the improvement of pharmacopoeia quality control, and the study of its metabolic pathways and mechanisms.

[0004] Ultra-high performance liquid phase high-resolution mass spectrometry was used to analyze the blood components that entered the body after Codonopsis pilosula was used to treat diseases, and the blood components were identified based on the secondary mass spectrum of the quasi-molecular ions and characteristic fragment ions. These compounds may be substances that produce pharmacological effects of Codonopsis pilosula in the body.

[0005] However, the entire process lacks information on the spatial dimension of the distribution of blood-entering components in plants, and lacks the distribution and positioning of substances in plant tissues and relative content determination after entering the body.

[0006] The types, contents and spatial distribution of components entering the blood have not been precisely measured. The material basis behind the biological effects is explained from the three dimensions of qualitative, quantitative and positional. The omics information is expanded from two-dimensional to three-dimensional level, and the metabolites are further confirmed from the spatial dimension. Summary of the invention

[0007] The present invention is to solve the above problems. The present invention provides a mass spectrometry imaging method for blood-entering pharmacological substances in Codonopsis pilosula roots and its application. The components that enter the blood and tissues after administration of Codonopsis pilosula are accurately located and analyzed in the roots of Codonopsis pilosula, revealing the specific distribution of effective components in the roots of Codonopsis pilosula, improving the exploration of the drug administration mechanism of chemical components of Codonopsis pilosula, and visualizing the active components that play a role. It is closely related to the extraction and separation of active substances in Codonopsis pilosula, improving the quality control of pharmacopoeia, and its metabolic pathway and mechanism research. The synthetic pathway and distribution location of Codonopsis pilosula are understood to the greatest extent by people, providing experience for clinical treatment.

[0008] To achieve the above object, the present invention provides the following technical solution: a mass spectrometry imaging method and application of blood-entering pharmacodynamic substances of Codonopsis root, the method comprising the following steps:

[0009] S1. Pharmacodynamics of Codonopsis pilosula in treating diseases and serum pharmacochemical analysis

[0010] (1) Establishment of animal model: Taking spleen deficiency model as an example

[0011] The experimental animals were kept in an environment with a temperature of about 20℃ and a relative humidity of 50±5%, with regular ventilation and sufficient space. The basic feed and drinking water were not limited. They were adaptively fed for 7 days before the start of the experiment. 72 rats were randomly divided into a normal control group (Control) of 12, a model control group (Model) of 12, a low-dose Codonopsis pilosula (CP-L) group of 12, a medium-dose Codonopsis pilosula (CP-M) group of 12, a high-dose Codonopsis pilosula (CP-H) group of 12, and a positive group of Sijunzi Decoction (SJZT) of 12.

[0012] (2) Serum drug chemistry analysis: sample processing

[0013] Take 150 μL of Codonopsis pilosula alcohol extract sample, put it in a 1.5 mL EP tube, and vacuum dry it; add 300 μL of 40% methanol aqueous solution to dissolve it; vortex mix for 30 seconds; centrifuge for 15 minutes (16000g, 4°C), and take the supernatant.

[0014] Take an appropriate amount of serum sample, add methanol, vortex mix for 60s, let stand at -20℃ for 30min, centrifuge at 16000g and 4℃ for 20min, take the supernatant and vacuum dry, add 100μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g and 4℃ for 15min, take the supernatant.

[0015] Take an appropriate amount of serum sample, add codonopsis pilosula extract, add methanol, vortex mix for 60s, let stand at 20℃ for 30min, centrifuge at 16000g at 4℃ for 20min, take the supernatant and vacuum dry, add 100μL 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g at 4℃ for 15min, take the supernatant.

[0016] (3) Chromatography-mass spectrometry analysis

[0017] The samples were separated using a Vanquish UHPLC (Thermo Scientific, Waltham, MA) ultra-high performance liquid chromatography system combined with an ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; the column temperature was 35° C.; the flow rate was 0.3 mL / min; the mobile phase composition was A 0.1% formic acid aqueous solution, B 0.1% formic acid acetonitrile solution; and the gradient elution was performed according to the table below.

[0018] Table 1 - Gradient elution schedule

[0019]

[0020] The Q-Exactive HFX mass spectrometer was used to collect the primary and secondary spectra of the samples. The Q-Exactive HFX mass spectrometer sample was connected to the UHPLC system, and the electrospray ionization (ESI) positive and negative ion modes were used for mass spectrometry acquisition. The spray voltage was 3800V (ESI+) / 3500V (ESI-), the sheath gas pressure was 45arb, the auxiliary gas pressure was 20arb, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃; the detection mode was full scan / data-dependent secondary scan (Full-MS / dd-MS2) mode, the primary and secondary resolutions were 60000 and 15000, respectively, the top 10MS1 ions obtained MS / MS spectra, and the collision energies (CEs) used step-normalized energy levels of 20, 40, and 60; the primary mass-to-charge ratio scanning range was 90-1300.

[0021] (4) Serum sample testing and analysis

[0022] 6 μL of blank group samples, drug group samples, blank group + codonopsis pilosula samples, and 2 μL of codonopsis pilosula solution were accurately pipetted for LC-MS injection analysis. Each batch of blank group samples and drug group samples were injected once, the blank group + codonopsis pilosula samples were injected 3 times, and the codonopsis pilosula samples were injected 5 times.

[0023] (5) Data processing

[0024] Import the raw data file in .raw format into proteoWizard and convert it into .mzXML format. Use XCMS software for peak alignment, retention time correction and peak extraction. Use the database to identify the compound. The primary mass error is less than 25ppm, and the secondary fragmentation spectrum matching score is greater than 0.7. The higher the score, the higher the spectrum similarity. It is generally believed that the identification result is reliable if it is above 0.7.

[0025] S2. Study on the distribution of blood-entering components in traditional Chinese medicine

[0026] The fresh roots of Codonopsis pilosula were washed and the main roots and lateral roots were separated and placed in a -80°C refrigerator until used for section examination.

[0027] Turn on the Thermo CRYOSTARNX50 cryostat and lower the temperature of the freezer to -10°C; take the tissue out of the -80°C freezer and place it in the freezer for 3 hours to equilibrate; fix the sample on the locatable sample head with embedding solution, adjust the angle and orientation of the sample, and slice according to the instructions of the slicer; the slice thickness used for mass spectrometry imaging is 14 μm; transfer the slice to the precooled ITO conductive slide with a precooled brush and dry for 20 minutes; if the sliced ​​sample is not tested immediately, store the ITO conductive slide at -80°C.

[0028] After marking the sample information on the ITO glass slide with cut tissue, turn on the circulating spray matrix spraying device (SunCollect, SunChrom) for matrix spraying. The matrix spraying information is as shown in the following table (if the ITO tissue slice is taken out from the -80°C storage refrigerator, it needs to be dried for 20 minutes before matrix spraying), the nozzle temperature is room temperature, the spraying speed is 1000mm / min, the flow rate of each layer of spraying changes in a gradient, ranging from 10-60μL / min, and 16 layers are sprayed.

[0029] Table 2 - Matrix Spraying Information

[0030]

[0031] In the mass spectrometry imaging experiment, the AP-MALDI ion source scans the sample on the slide line by line, and then saves the mass spectrometry data obtained in each line as a RAW file. The mass spectrometry spatial image is reconstructed and visualized based on the MSI processing algorithm, and the software system platform (http: / / www.biodeep.cn) automatically processes the MSI data. First, the RAW raw data file is converted into the universal mzML open source file format, and then peak detection and peak quantification are performed. The TrIQ algorithm is used to analyze and identify the background noise and signal correction for the detection intensity of the mass spectrometry ion signal. The data is reduced in dimension and the background signal is subtracted before downstream data analysis.

[0032] Based on an unsupervised algorithm, the UMAP Manifold method was used to perform dimensionality reduction and cluster analysis on high-dimensional mass spectrometry imaging spatial data. The phenograph clustering algorithm was then used to obtain different typing areas and features on the slices. The specific differential metabolites between different typings were found based on the spatial analysis data, and the biological mechanism was explained based on KEGG metabolic pathway analysis.

[0033] Preferably, in step S1, the rats in the blank group are gavaged with 2 mL of normal saline at 7 am every day, and 0.5 mg / kg of normal saline is injected intraperitoneally at 7 pm, the rats in the model group are gavaged with 2 mL of normal saline at 7 am every day, and 0.5 mg / kg of reserpine is injected intraperitoneally at 7 pm, the rats in the Codonopsis group are gavaged with Codonopsis pilosula alcohol extract at 7 am every day, the low dose is gavaged with 2 g / kg, the medium dose is gavaged with 4 g / kg, the high dose is gavaged with 8 g / kg, and 0.5 mg / kg of reserpine is injected intraperitoneally at 7 pm, and the Sijunzi Decoction positive group is gavaged with 20 g / kg of Sijunzi Decoction at 7 am every day, and 0.5 mg / kg of reserpine is injected intraperitoneally at 7 pm, for 14 consecutive days.

[0034] Preferably, in step S2, before performing multivariate statistical analysis on the metabolomics data, the data needs to be properly weighted, i.e., standardized (Scaling) processing. Currently, the commonly used data standardization methods in metabolomics research include centralization, adaptive scaling and Pareto scaling. This analysis uses adaptive scaling for the data to obtain more reliable and more intuitive results.

[0035] Preferably, in step S2, the original LC-MS / MS data is processed by Compound Discoverer3.1, and metabolite identification uses m / z Cloud and chemspider databases, including the Human Metabolome Database (MDB), Kyoto Encyclopedia of Genes and Genomes (KEGG) and LipidMaps. All values ​​are expressed as mean ± standard error. SPSS19.0 software is used for T test (P<0.05) statistical analysis, and SIMCA 14. software is used for partial least squares discriminant analysis (PLS-DA). Usually, the variable importance (VIP) value is greater than 1, indicating that the variable has a significant effect on the classification of the sample. In summary, VIP>1 and P value<0.05 are used to screen differential metabolites for identification of differentially expressed metabolites.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention uses mass spectrometry imaging technology to accurately identify and locate the differential distribution of multiple pharmacological substances in different tissues of plants, and quickly conducts deep mass spectrometry imaging analysis on target micro-regional tissues to obtain the types and content information of components entering the blood, thereby achieving the purpose of clarifying the distribution of pharmacological substances;

[0038] 2. The present invention incorporates spatial metabolomics, which can complete the exploration of the entire mechanism of action of Codonopsis pilosula in treating diseases, including the pharmacodynamics study of Codonopsis pilosula, and the search for the effective substance components and their spatial distribution in the roots of Codonopsis pilosula. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the overall experimental process diagram of the present invention;

[0040] Figure 2 is the spatial metabolic flow chart of the present invention;

[0041] Figure 3 The optical photograph of Codonopsis pilosula of the present invention, stained with safranin fast green, and the total ion current diagram of mass spectrometry imaging;

[0042] Figure 4 This is a mass spectrometry image of the main components of the root of Codonopsis pilosula of the present invention;

[0043] Figure 5 The mass spectrometry imaging diagram of the blood-entering components of the present invention and the content comparison diagram of different partitions;

[0044] Figure 6 This is a mass spectrum comparison imaging diagram of the main root and branch roots of Codonopsis pilosula of the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] like Figure 1-Figure 6 As shown, a mass spectrometry imaging method and application of blood-entering pharmacodynamic substances of Codonopsis root traditional Chinese medicine, the method comprises the following steps:

[0047] S1. Pharmacodynamics of Codonopsis pilosula in treating diseases and serum pharmacochemical analysis

[0048] (1) Establishment of animal model: Taking spleen deficiency model as an example

[0049] The experimental animals were kept in an environment with a temperature of about 20℃ and a relative humidity of 50±5%, with regular ventilation and sufficient space. The basic feed and drinking water were not limited. They were adaptively fed for 7 days before the start of the experiment. 72 rats were randomly divided into a normal control group (Control) of 12, a model control group (Model) of 12, a low-dose Codonopsis pilosula (CP-L) group of 12, a medium-dose Codonopsis pilosula (CP-M) group of 12, a high-dose Codonopsis pilosula (CP-H) group of 12, and a positive group of Sijunzi Decoction (SJZT) of 12.

[0050] (2) Serum drug chemistry analysis: sample processing

[0051] Take 150 μL of Codonopsis pilosula alcohol extract sample, put it in a 1.5 mL EP tube, and vacuum dry it; add 300 μL of 40% methanol aqueous solution to dissolve it; vortex mix for 30 seconds; centrifuge for 15 minutes (16000g, 4°C), and take the supernatant.

[0052] Take an appropriate amount of serum sample, add methanol, vortex mix for 60s, let stand at -20℃ for 30min, centrifuge at 16000g and 4℃ for 20min, take the supernatant and vacuum dry, add 100μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g and 4℃ for 15min, take the supernatant.

[0053] Take an appropriate amount of serum sample, add codonopsis pilosula extract, add methanol, vortex mix for 60s, let stand at 20℃ for 30min, centrifuge at 16000g at 4℃ for 20min, take the supernatant and vacuum dry, add 100μL 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g at 4℃ for 15min, take the supernatant.

[0054] (3) Chromatography-mass spectrometry analysis

[0055] The samples were separated using a Vanquish UHPLC (Thermo Scientific, Waltham, MA) ultra-high performance liquid chromatography system combined with an ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; the column temperature was 35° C.; the flow rate was 0.3 mL / min; the mobile phase composition was A 0.1% formic acid aqueous solution, B 0.1% formic acid acetonitrile solution; and the gradient elution was performed according to the table below.

[0056] Table 1 - Gradient elution schedule

[0057]

[0058] The Q-Exactive HFX mass spectrometer was used to collect the primary and secondary spectra of the samples. The Q-Exactive HFX mass spectrometer sample was connected to the UHPLC system, and the electrospray ionization (ESI) positive and negative ion modes were used for mass spectrometry acquisition. The spray voltage was 3800V (ESI+) / 3500V (ESI-), the sheath gas pressure was 45arb, the auxiliary gas pressure was 20arb, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃; the detection mode was full scan / data-dependent secondary scan (Full-MS / dd-MS2) mode, the primary and secondary resolutions were 60000 and 15000, respectively, the top 10MS1 ions obtained MS / MS spectra, and the collision energies (CEs) used step-normalized energy levels of 20, 40, and 60; the primary mass-to-charge ratio scanning range was 90-1300.

[0059] (4) Serum sample testing and analysis

[0060] 6 μL of blank group samples, drug group samples, blank group + codonopsis pilosula samples, and 2 μL of codonopsis pilosula solution were accurately pipetted for LC-MS injection analysis. Each batch of blank group samples and drug group samples were injected once, the blank group + codonopsis pilosula samples were injected 3 times, and the codonopsis pilosula samples were injected 5 times.

[0061] (5) Data processing

[0062] Import the raw data file in .raw format into proteoWizard and convert it into .mzXML format. Use XCMS software for peak alignment, retention time correction and peak extraction. Use the database to identify the compound. The primary mass error is less than 25ppm, and the secondary fragmentation spectrum matching score is greater than 0.7. The higher the score, the higher the spectrum similarity. It is generally believed that the identification result is reliable if it is above 0.7.

[0063] S2. Study on the distribution of blood-entering components in traditional Chinese medicine

[0064] The fresh roots of Codonopsis pilosula were washed and the main roots and lateral roots were separated and placed in a -80°C refrigerator until used for section examination.

[0065] Turn on the Thermo CRYOSTARNX50 cryostat and lower the temperature of the freezer to -10°C; take the tissue out of the -80°C freezer and place it in the freezer for 3 hours to equilibrate; fix the sample on the locatable sample head with embedding solution, adjust the angle and orientation of the sample, and slice according to the instructions of the slicer; the slice thickness used for mass spectrometry imaging is 14 μm; transfer the slice to the precooled ITO conductive slide with a precooled brush and dry for 20 minutes; if the sliced ​​sample is not tested immediately, store the ITO conductive slide at -80°C.

[0066] After marking the sample information on the ITO glass slide with cut tissue, turn on the circulating spray matrix spraying device (SunCollect, SunChrom) for matrix spraying. The matrix spraying information is as shown in the following table (if the ITO tissue slice is taken out from the -80°C storage refrigerator, it needs to be dried for 20 minutes before matrix spraying), the nozzle temperature is room temperature, the spraying speed is 1000mm / min, the flow rate of each layer of spraying changes in a gradient, ranging from 10-60μL / min, and 16 layers are sprayed.

[0067] Table 2 - Matrix Spraying Information

[0068]

[0069] In the mass spectrometry imaging experiment, the AP-MALDI ion source scans the sample on the slide line by line, and then saves the mass spectrometry data obtained in each line as a RAW file. The mass spectrometry spatial image is reconstructed and visualized based on the MSI processing algorithm, and the software system platform (http: / / www.biodeep.cn) automatically processes the MSI data. First, the RAW raw data file is converted into the universal mzML open source file format, and then peak detection and peak quantification are performed. The TrIQ algorithm is used to analyze and identify the background noise and signal correction for the detection intensity of the mass spectrometry ion signal. The data is reduced in dimension and the background signal is subtracted before downstream data analysis.

[0070] Based on an unsupervised algorithm, the UMAP Manifold method was used to perform dimensionality reduction and cluster analysis on high-dimensional mass spectrometry imaging spatial data. The phenograph clustering algorithm was then used to obtain different typing areas and features on the slices. The specific differential metabolites between different typings were found based on the spatial analysis data, and the biological mechanism was explained based on KEGG metabolic pathway analysis.

[0071] In this embodiment: in step S1, the rats in the blank group were gavaged with 2 mL of normal saline at 7 am every day, and 0.5 mg / kg of normal saline was intraperitoneally injected at 7 pm, the rats in the model group were gavaged with 2 mL of normal saline at 7 am every day, and 0.5 mg / kg of reserpine was intraperitoneally injected at 7 pm, the rats in the Codonopsis group were gavaged with Codonopsis pilosula alcohol extract at 7 am every day, the low dose was gavage 2 g / kg, the medium dose was gavage 4 g / kg, the high dose was gavage 8 g / kg, and 0.5 mg / kg of reserpine was intraperitoneally injected at 7 pm, and the Sijunzi Decoction positive group was gavaged with 20 g / kg of Sijunzi Decoction at 7 am every day, and 0.5 mg / kg of reserpine was intraperitoneally injected at 7 pm, for 14 consecutive days.

[0072] In this embodiment: In step S2, before performing multivariate statistical analysis on the metabolomics data, the data needs to be properly weighted, i.e., standardized (Scaling) processing. Currently, the commonly used data standardization methods in metabolomics research include centralization, adaptive scaling and Pareto scaling. This analysis uses adaptive scaling for the data to obtain more reliable and more intuitive results.

[0073] In this embodiment: in step S2, the original LC-MS / MS data is processed by Compound Discoverer3.1, and metabolite identification uses m / z Cloud and chemspider databases, including the Human Metabolome Database (MDB), Kyoto Encyclopedia of Genes and Genomes (KEGG) and LipidMaps. All values ​​are expressed as mean ± standard error. SPSS19.0 software is used for T test (P<0.05) statistical analysis, and SIMCA 14. software is used for partial least squares discriminant analysis (PLS-DA). Generally, a variable importance (VIP) value greater than 1 indicates that the variable has a significant effect on the classification of the sample. In summary, VIP>1 and P value<0.05 are used to screen differential metabolites for identification of differentially expressed metabolites.

[0074] In summary, compared with the existing technology, the present invention uses mass spectrometry imaging technology to accurately identify and locate the differential distribution of multiple pharmacological substances in different tissues of plants, and quickly performs deep mass spectrometry imaging analysis on target micro-regional tissues to obtain the types and content information of components entering the blood, so as to achieve the purpose of clarifying the distribution of pharmacological substances. This technology incorporates spatial metabolomics to complete the exploration of the entire mechanism of action of Codonopsis pilosula in treating diseases, including the pharmacological study of Codonopsis pilosula, and the search for pharmacological substances and their spatial distribution in Codonopsis pilosula roots.

[0075] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a 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.

[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A mass spectrometry imaging method and application of blood-entering pharmacodynamic substances of Codonopsis pilosula root, characterized in that: The method comprises the following steps: S1. Pharmacodynamics of Codonopsis pilosula in treating diseases and serum pharmacochemical analysis (1) Establishment of animal model: Taking spleen deficiency model as an example The experimental animals were kept in an environment with a temperature of about 20℃ and a relative humidity of 50±5%, with regular ventilation and sufficient space. The basic feed and drinking water were not limited. They were adaptively fed for 7 days before the start of the experiment. 72 rats were randomly divided into a normal control group (Control) of 12, a model control group (Model) of 12, a low-dose Codonopsis pilosula (CP-L) group of 12, a medium-dose Codonopsis pilosula (CP-M) group of 12, a high-dose Codonopsis pilosula (CP-H) group of 12, and a positive group of Sijunzi Decoction (SJZT) of 12. (2) Serum drug chemistry analysis: sample processing Take 150 μL of Codonopsis pilosula alcohol extract sample, put it in a 1.5 mL EP tube, and vacuum dry it; add 300 μL of 40% methanol aqueous solution to dissolve it; vortex mix for 30 seconds; centrifuge for 15 minutes (16000g, 4°C), and take the supernatant. Take an appropriate amount of serum sample, add methanol, vortex mix for 60s, let stand at -20℃ for 30min, centrifuge at 16000g and 4℃ for 20min, take the supernatant and vacuum dry, add 100μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g and 4℃ for 15min, take the supernatant. Take an appropriate amount of serum sample, add codonopsis pilosula extract, add methanol, vortex mix for 60s, let stand at 20℃ for 30min, centrifuge at 16000g at 4℃ for 20min, take the supernatant and vacuum dry, add 100μL 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g at 4℃ for 15min, take the supernatant. (3) Chromatography-mass spectrometry analysis The samples were separated using a Vanquish UHPLC (Thermo Scientific, Waltham, MA) ultra-high performance liquid chromatography system combined with an ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; the column temperature was 35° C.; the flow rate was 0.3 mL / min; the mobile phase composition was A 0.1% formic acid aqueous solution, B 0.1% formic acid acetonitrile solution; and the gradient elution was performed according to the table below. Table 1 - Gradient elution schedule The Q-Exactive HFX mass spectrometer was used to collect the primary and secondary spectra of the samples. The Q-Exactive HFX mass spectrometer sample was connected to the UHPLC system, and the electrospray ionization (ESI) positive and negative ion modes were used for mass spectrometry acquisition. The spray voltage was 3800V (ESI+) / 3500V (ESI-), the sheath gas pressure was 45arb, the auxiliary gas pressure was 20arb, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃; the detection mode was full scan / data-dependent secondary scan (Full-MS / dd-MS2) mode, the primary and secondary resolutions were 60000 and 15000, respectively, the top 10MS1 ions obtained MS / MS spectra, and the collision energies (CEs) used step-normalized energy levels of 20, 40, and 60; the primary mass-to-charge ratio scanning range was 90-1300. (4) Serum sample testing and analysis 6 μL of blank group samples, drug group samples, blank group + codonopsis pilosula samples, and 2 μL of codonopsis pilosula solution were accurately pipetted for LC-MS injection analysis. Each batch of blank group samples and drug group samples were injected once, the blank group + codonopsis pilosula samples were injected 3 times, and the codonopsis pilosula samples were injected 5 times. (5) Data processing Import the raw data file in .raw format into proteoWizard and convert it into .mzXML format. Use XCMS software for peak alignment, retention time correction and peak extraction. Use the database to identify the compound. The primary mass error is less than 25ppm, and the secondary fragmentation spectrum matching score is greater than 0.

7. The higher the score, the higher the spectrum similarity. It is generally believed that the identification result is reliable if it is above 0.

7. S2. Study on the distribution of blood-entering components in traditional Chinese medicine The fresh roots of Codonopsis pilosula were washed and the main roots and lateral roots were separated and placed in a -80°C refrigerator until used for section examination. Turn on the Thermo CRYOSTARNX50 cryostat and lower the temperature of the freezer to -10°C; take the tissue out of the -80°C freezer and place it in the freezer for 3 hours to equilibrate; fix the sample on the locatable sample head with embedding solution, adjust the angle and orientation of the sample, and slice according to the instructions of the slicer; the slice thickness used for mass spectrometry imaging is 14 μm; transfer the slice to the precooled ITO conductive slide with a precooled brush and dry for 20 minutes; if the sliced ​​sample is not tested immediately, store the ITO conductive slide at -80°C. After marking the sample information on the ITO glass slide with cut tissue, turn on the circulating spray matrix spraying device (SunCollect, SunChrom) for matrix spraying. The matrix spraying information is as shown in the following table (if the ITO tissue slice is taken out from the -80°C storage refrigerator, it needs to be dried for 20 minutes before matrix spraying), the nozzle temperature is room temperature, the spraying speed is 1000mm / min, the flow rate of each layer of spraying changes in a gradient, ranging from 10-60μL / min, and 16 layers are sprayed. Table 2 - Matrix Spraying Information In the mass spectrometry imaging experiment, the AP-MALDI ion source scans the sample on the slide line by line, and then saves the mass spectrometry data obtained in each line as a RAW file. The mass spectrometry spatial image is reconstructed and visualized based on the MSI processing algorithm, and the software system platform (http: / / www.biodeep.cn) automatically processes the MSI data. First, the RAW raw data file is converted into the universal mzML open source file format, and then peak detection and peak quantification are performed. The TrIQ algorithm is used to analyze and identify the background noise and signal correction for the detection intensity of the mass spectrometry ion signal. The data is reduced in dimension and the background signal is subtracted before downstream data analysis. Based on an unsupervised algorithm, the UMAP Manifold method was used to perform dimensionality reduction and cluster analysis on high-dimensional mass spectrometry imaging spatial data. The phenograph clustering algorithm was then used to obtain different typing areas and features on the slices. The specific differential metabolites between different typings were found based on the spatial analysis data, and the biological mechanism was explained based on KEGG metabolic pathway analysis.

2. The method and application of mass spectrometry imaging of blood-active substances of Codonopsis root according to claim 1, characterized in that: In step S1, the rats in the blank group were gavaged with 2 mL of normal saline at 7 am every day, and intraperitoneally injected with 0.5 mg / kg of normal saline at 7 pm every day, the rats in the model group were gavaged with 2 mL of normal saline at 7 am every day, and intraperitoneally injected with 0.5 mg / kg of reserpine at 7 pm every day, the rats in the Codonopsis group were gavaged with Codonopsis pilosula alcohol extract at 7 am every day, the low dose was gavage 2 g / kg, the medium dose was gavage 4 g / kg, the high dose was gavage 8 g / kg, and the rats were intraperitoneally injected with 0.5 mg / kg of reserpine at 7 pm, and the Sijunzi Decoction positive group was gavaged with 20 g / kg of Sijunzi Decoction at 7 am every day, and intraperitoneally injected with 0.5 mg / kg of reserpine at 7 pm, for 14 consecutive days.

3. The method and application of mass spectrometry imaging of blood-active substances of Codonopsis root according to claim 1, characterized in that: In step S2, before performing multivariate statistical analysis on the metabolomics data, the data needs to be properly weighted, i.e., standardized (Scaling) processing. Currently, the commonly used data standardization methods in metabolomics research include central processing, adaptive scaling, and Pareto scaling. This analysis uses adaptive scaling for the data to obtain more reliable and more intuitive results.

4. The method and application of mass spectrometry imaging of blood-active substances of Codonopsis root according to claim 1, characterized in that: In step S2, the raw LC-MS / MS data were processed by Compound Discoverer 3.1, and metabolite identification was performed using m / z Cloud and chemspider databases, including the Human Metabolome Database (MDB), Kyoto Encyclopedia of Genes and Genomes (KEGG), and LipidMaps. All values ​​were expressed as mean ± standard error. SPSS19.0 software was used for T test (P<0.05) statistical analysis, and SIMCA 14. software was used for partial least squares discriminant analysis (PLS-DA). Generally, a variable importance (VIP) value greater than 1 indicates that the variable has a significant effect on the classification of the sample. In summary, VIP>1 and P value<0.05 were used to screen differential metabolites for identification of differentially expressed metabolites.

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