CN110794074A - Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method - Google Patents
Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method Download PDFInfo
- Publication number
- CN110794074A CN110794074A CN201911128114.3A CN201911128114A CN110794074A CN 110794074 A CN110794074 A CN 110794074A CN 201911128114 A CN201911128114 A CN 201911128114A CN 110794074 A CN110794074 A CN 110794074A
- Authority
- CN
- China
- Prior art keywords
- cold
- metabolic pathway
- rats
- analysis
- urine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a Chinese angelica Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and a research method, wherein the method comprises the following steps: adopting ultra performance liquid chromatography tandem mass spectrometry to detect and analyze endogenous metabolites which are induced by an ice water bath and adrenalin to form congealing cold blood stasis syndrome and angelica sinensis Sini soup stem prognosis and change at different time points to obtain a fingerprint; screening and identifying 21 remarkably changed metabolites from a plurality of variables by using a multivariate statistical analysis method; eight metabolic pathways related to different development stages of the blood stasis syndrome due to congealing cold are enriched through a metaboanalyst open source online metabonomics analysis website. The invention utilizes a dynamic analysis method to research the metabolic changes related to the occurrence and development process of the congealing cold and blood stasis syndrome and the treatment process of the angelica sinica decoction, is helpful for disclosing the occurrence and development mechanism of diseases at different time points and clarifying an abnormal metabolic network regulated and controlled by medicaments in the treatment process.
Description
Technical Field
The invention relates to the technical field of medical analysis. More particularly, the invention relates to a Chinese angelica Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and a research method.
Background
Metabonomics (metabonomics) has the characteristic of 'whole-dynamic-comprehensive', which is consistent with the pathogenesis process of complex diseases and the onset characteristic of traditional Chinese medicines. Metabolomics is a new technology developed in recent years for qualitative and quantitative analysis of changes in dynamic metabolites of biological systems, and is commonly used for studying the changes of endogenous small molecule metabolites to external stimuli (including environmental, physiological and pathological stimuli). The metabonomic research process generally comprises the processes of biological sample collection, pretreatment, sample analysis, data processing, marker identification, biological meaning explanation and the like. The technology mainly comprises Nuclear Magnetic Resonance (NMR), gas chromatography-mass spectrometry (GS/MS) combined technology, liquid chromatography-mass spectrometry (LC/MS) combined technology and the like. In recent years, high throughput metabolomic analysis has often been used to describe the metabolic profile of biological samples. Because the ultra-high performance liquid phase-quadrupole flight mass spectrometry (UPLC-Q-TOF/MS) can rapidly acquire a large amount of accurate mass number information and has the characteristics of high resolution and high sensitivity, the technology is widely used for dynamic analysis of metabolic changes in organisms and mechanism research of drug therapy. In conjunction with multivariate statistical analysis, the metabolic profiles of the treatment, disease model and blank groups can be visualized, the changes in metabolic profiles analyzed, and endogenous metabolites that are significantly different between groups identified.
The syndrome of congealing cold and blood stasis (BSS) is defined as poor blood circulation, reduced blood flow to the body or thick blood, is a common chronic and comprehensive disease in traditional Chinese medicine, and is the pathological basis of many serious and incurable diseases, such as coronary heart disease, infertility and cancer. Therefore, the search for the labeled metabolites capable of finding the BSS in the occurrence and development process and the elucidation of the action mechanism of the medicament with the definite curative effect have important significance for the early diagnosis and treatment of the BSS and also have very important significance and urgency for improving the health level of people in China. For a long time, researchers in medicine have been dedicated to research on pathogenesis of congealing cold and blood stasis syndrome and therapeutic drugs. The pathogenesis of BSS is complex, and is a dynamic process involving multiple links, factors and networks. If the disease cannot intervene in time, it will progress to a more serious disease. Therefore, the study of the pathogenesis of BSS would facilitate early intervention of the disease. However, the pathogenesis of BSS throughout its development has not been elucidated.
In the research of traditional Chinese medicine, the traditional Chinese medicine draws wide attention to the treatment of BSS. The traditional Chinese medicine has unique advantages in the aspect of treating complex diseases. In recent years, TCM has been widely applied worldwide with its unique theoretical system and long-standing clinical practice. Dang Gui Sixin Tang (DSD) is a classic Chinese medicine prescription widely used for treating diseases such as congealing cold and blood stasis. The research shows that it has the function of promoting blood circulation obviously. The traditional Chinese medicine mainly comprises seven traditional Chinese medicines, namely angelica sinensis, cassia twig, white paeony root, asarum, ricepaper pith, liquorice and Chinese date. Since the development of BSS is a slow and dynamic process, the mechanism of DSD intervention in the development of BSS has not been studied.
Disclosure of Invention
The invention aims to provide a Chinese angelica decoction for resisting cold, coagulating blood stasis and differentiating metabolite metabolism pathway and a research method thereof, and the research method utilizes a dynamic analysis method to research related metabolic changes in the BSS occurrence and development process and the DSD treatment process, is helpful to disclose the occurrence and development mechanism of diseases at different time points and clarify the abnormal metabolic network regulated by drugs in the treatment process.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for researching a cold coagulation blood stasis resistant differential metabolite metabolism pathway of Dang Gui Sini Tang, comprising:
adopting ultra performance liquid chromatography tandem mass spectrometry to detect and analyze endogenous metabolites which are induced by an ice water bath and adrenalin to form congealing cold blood stasis syndrome and angelica sinensis Sini soup stem prognosis and change at different time points to obtain a fingerprint;
utilizing a multivariate variable statistical analysis method to screen and identify twenty-one metabolites with significant changes from a plurality of variables as differential metabolites potentially related to the occurrence and development of congealing cold blood stasis;
eight metabolic pathways related to different development stages of the blood stasis syndrome due to congealing cold are enriched through a metaboanalyst open source online metabonomics analysis website.
Preferably, in the angelica sinensis sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and the research method, the specific steps for obtaining the angelica sinensis sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway are as follows:
(1) preparation of Angelica sinensis Sini decoction
Placing 12g of angelica sinensis, 9g of cassia twig, 9g of white paeony root, 3g of asarum, 6g of ricepaper pith, 6g of honey-fried licorice root and 12g of Chinese date in a 1000mL round-bottom flask, firstly soaking the components in distilled water with the weight being 8 times of the weight of the components for 1h, then carrying out heating reflux extraction at the normal pressure and the temperature of 100 ℃ for 2h, collecting a first extracting solution, and keeping filter residues;
adding water with the weight 6 times of that of the components into a 1000mL round-bottom flask, continuously heating and refluxing the filter residue for extraction for 2 hours, and collecting a second extracting solution;
mixing the first extractive solution and the second extractive solution, and filtering with 200 mesh gauze;
placing the filtered filtrate in a vacuum rotary evaporator, and concentrating at 60 deg.C until the concentration of the final concentrated solution is 2.0g/mL to obtain radix Angelicae sinensis Sini decoction;
(2) determination of animal models
After 24 female rats are adaptively fed for one week, randomly dividing the rats into a blank group, a model group and a DSD treatment group according to a random digital method, wherein each group comprises 8 rats, the rats in the model group and the DSD treatment group are placed in a cold water bath at 0-2 ℃ for swimming, and when the rats have slow response, weak breathing and stiff legs and are drowned, the rats are taken out and stimulated once a day for 14 days; according to the parallel control principle, the rats in the blank group are placed in a water bath at 35-37 ℃ for swimming for 5-10min, and swim for 1 time every day for 14 days continuously; when the experiment was performed by day 10, rats in the model group and the DSD treatment group were injected subcutaneously with epinephrine hydrochloride twice at 4 hour intervals at a dose of 0.8 mg/kg; 2h after the first injection, the rats in the model group and the DSD treatment group are placed in a cold water bath at 0-2 ℃ for swimming for 5min, and the rats in the blank group are placed in a water bath at 35-37 ℃ for swimming for 5 min;
the dosing method of this study was: during the modeling period, the DSD treatment group performs intragastric administration on the angelica sinensis Sini decoction every day with the dose of 1.8mg/g, and the blank group and the model group perform intragastric administration on distilled water with equal dose in parallel once a day for 14 days;
(3) collection, preservation and pretreatment of rat urine
On days 0, 5, 10, 14 during animal molding, rats per group were collected at night 20: 00 to morning 8: 00, collecting urine samples in the time period by using 5mL centrifuge tubes respectively, and burying each centrifuge tube in an ice box to enable each urine sample to be at 0-4 ℃;
after an equal amount of 600 mu L urine sample is taken out from each sample, adding sodium azide with the mass concentration of 0.1 percent into each urine sample, and then freezing and storing each centrifugal tube at the temperature of minus 80 ℃;
unfreezing each urine sample at room temperature, diluting each urine sample with 600 mu L of deionized water, centrifuging each urine sample to remove precipitates, filtering each urine sample through a 0.22 micron polyvinylidene fluoride filter membrane, respectively filling the urine samples into sample injection vials, and then taking 10 mu L of urine sample from each sample and mixing the urine sample in 1 new centrifugal tube to prepare a quality control QC sample;
(4) ultra-high performance liquid chromatography tandem mass spectrometry detection analysis of urine sample
(5) Data pre-processing
Performing denoising, mass spectrum peak extraction, peak arrangement, peak alignment and normalization processing on the acquired ultra performance liquid chromatography and mass spectrum detection data by adopting Masslynx software to obtain the peak height or peak area and retention time of each peak;
(6) metabolic profile differences and dynamics analysis using multivariate variables statistics
Importing the preprocessed data into SIMCA-P14.1 software, preprocessing the data by adopting a Par scaling method, then carrying out principal component analysis on blank groups, model groups and QC samples, carrying out PCA (principal component analysis) track analysis on the blank groups, the model groups and the DSD (differential motion detection) treatment group urine samples collected at different time points, and carrying out partial least square discriminant analysis on the blank groups, the model groups and the DSD treatment groups;
(7) screening for potential biomarkers
Establishing an orthogonal partial least square analysis model by using the detection data of the ultra performance liquid chromatography and the mass spectrum after the pretreatment of the blank group and the model group, screening differential metabolites between the two groups, and performing statistical processing by adopting SPSS16.0 software; the screening conditions for differential metabolites were: selecting a projection value of variable importance >1, wherein the difference has statistical significance between the two groups;
(8) identification of potential biomarkers
And (3) identifying potential biomarkers by searching public databases KEGG, HMDB and METLIN according to the accurate mass number and the secondary characteristic fragments corresponding to the variables, and comparing the potential biomarkers with a document which describes the identification mass spectrum information of the metabolites to finally identify twenty differential metabolites related to the occurrence and development of BSS: 1-methylnicotinamide, creatinine, N-methylhistidine, tyramine glucuronic acid, 1-methylguanine, 1-methyladenine, 5-methylcytosine, 3-hydroxyaminobenzoic acid, leucine-proline, 2-phenylacetamide, indoline, 2-methyl-1, 2,3, 4-tetrahydro-6, 7-isoquinolinediol, xanthurenic acid, glucuronic acid ester of 3-indolecarboxylic acid, hippuric acid, riboflavin, phenylacetylglycine, 5-methoxyindole acetate, corticosterone, allocholic acid, tetrahydrocorticosterone;
(9) metabolic pathway enrichment assay
Uploading the identified twenty-one differential metabolite to a metaboanalyst open source online metabonomics analysis website, inputting urine metabolite names of different days, standardizing the metabolite names, and selecting a KEGG database of rat species to enrich related pathways; carrying out enrichment analysis and topological analysis on the significantly influenced metabolic pathways in urine of BSS rats in different periods, and obtaining eight metabolic pathways by total enrichment: nicotinic acid and nicotinamide metabolic pathway, starch and sucrose metabolic pathway, histidine metabolic pathway, pentose and glucuronic acid metabolic pathway, tryptophan metabolic pathway, phenylalanine metabolic pathway, riboflavin metabolic pathway, and steroid hormone biosynthetic metabolic pathway.
Preferably, in the angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and the research method, the ultra-performance liquid chromatography detection adopts an ultra-performance liquid system of Waters company to carry out detection, and the HSST is3A chromatographic column with specification of 100mm multiplied by 2.1mm, fixed phase particle size of 1.8 μm, experimental column temperature of 25 ℃, mobile phase A water, B acetonitrile containing chromatographic formic acid, wherein, the volume fraction of the chromatographic formic acid is 0.1%, and the flow rate of the mobile phase is 0.4 mL/min-1The gradient elution procedure was: 1% of B,0-0.5 min; 1-10% of B,0.5-7 min; 10-50% of B,7-15 min; 50-100% B,15-17min, 100% B,17-19 min; 100-1% B,19-20 min; the temperature of the automatic sample introduction chamber was set to 4 ℃ and the sample introduction amount was 10. mu.L.
Preferably, in the angelica sinensis Sini-Rev decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolism pathway and the research method, mass spectrometry detection is performed by using a quadrupole-time-of-flight mass spectrometer of Waters company, the mass number range of the collected mass spectrum is 100-1000Da, and all data are collected in an electrospray ion ESI source positive ion mode, wherein the capillary tube voltage is 4.0kV, the spinal hole voltage is 35kV, and the extraction voltage is 4.0 kV; the ion source temperature is 120 ℃, and the desolventizing gas temperature is 350 ℃; taper hole air flow rate of 40 L.h-1Desolventizing flow rate of 800 L.h-1Data acquisition in the Centriod model, using LockSpray from WatersTMThe system carries out real-time correction, leucine-enkephalin is taken as a mass reference component, the flow rate of a reference solution is 20 mu L/min, 8 urine samples are analyzed in the sequence, and a QC sample is injected once.
Preferably, in the angelica sinensis sini decoction cold-resistant blood-coagulation-stasis-resisting differential metabolite metabolic pathway and the research method, main processing parameters of data preprocessing are as follows: the retention time is 0-20 min; the mass number range is 100-1000 Da; mass number tolerance 0.01; mass number window 0.05; noise removal degree 6, after data was processed through 80% filtering principle, the variable with blank value > 80% was removed.
Preferably, in the angelica sinensis sini decoction cold coagulation and blood stasis resistant differential metabolite metabolic pathway and the research method, when each urine sample is centrifuged to remove precipitates, the urine sample is firstly centrifuged at a low speed of 3500 rpm for 10 minutes, and then centrifuged at a high speed of 12000 rpm for 10 minutes.
The invention at least comprises the following beneficial effects:
according to the invention, the dynamic analysis is carried out on metabolites in urine samples of various groups of rats at different time points by using the ultra-high performance liquid chromatography-tandem mass spectrometry technology, and different metabolites related to BSS development are found.
The content change of the biological marker of BSS is discovered by a dynamic urine analysis technology, and the metabolic pathway of the differential urine metabolite is analyzed to obtain the metabolic pathway disturbed in the development of BSS.
According to the invention, the content change of the marker for the intervention of the DSD in BSS is found through analysis, and the metabolites regulated and controlled by the DSD when the DSD acts at different periods are determined.
According to the invention, through ROC analysis, BSS differential metabolites with higher diagnostic value are discovered.
The invention comprehensively and efficiently discloses the BSS generation and development mechanism and the DSD action mechanism from the overall level, and provides an exemplary research for the clarification of the urine metabonomics and the action mechanism of the effective components of the traditional Chinese medicine formula.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a urine metabolism fingerprint of a model group rat collected in a positive ion mode based on ultra performance liquid chromatography-tandem mass spectrometry at day 0;
FIG. 2 is a urine metabolism fingerprint of a model group rat collected in a positive ion mode based on ultra performance liquid chromatography-tandem mass spectrometry on day 5;
FIG. 3 is a urine metabolism fingerprint of a model group rat collected in the positive ion mode based on ultra performance liquid chromatography-tandem mass spectrometry on day 10;
FIG. 4 is a urine metabolism fingerprint of a model group rat collected in the positive ion mode based on ultra performance liquid chromatography-tandem mass spectrometry at day 14;
FIG. 5 is a three-dimensional PCA chart on days 0 (indicated by (A) in FIG. 5), 5 (indicated by (B) in FIG. 5), 10 (indicated by (C) in FIG. 5) and 14 (indicated by (D) in FIG. 5); in FIG. 5, C is a blank set, M is a model set, and QC is a quality control sample;
FIG. 6 is a graph of PCA trace analysis of rat urine on different days; in FIG. 6, C is blank group, M is model group, and DSD is DSD treatment group;
FIG. 7 is a graph of PLS-DA urine from rats at days 5, 10 and 14; in FIG. 7, C is blank, M is model, and Q is DSD treatment;
FIG. 8 is an S-plot of the OPLS-DA model of rat urine at day 5, 10, 14;
FIG. 9 is an enrichment map of metabolic pathways constructed on-line with the metaboanalyst platform;
FIG. 10 is a summary of metabolic pathways; metabolites in the dotted line bold circle in FIG. 10 are lower difference metabolites; FIG. 10 shows the upward arrow indicating that the metabolites are up-regulated in the model group relative to the normal group; the reverse is true when the lower part is downward; p x <0.05, P x <0.01 compared to normal group.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
A Chinese angelica root Sini decoction differential metabolite metabolic pathway for treating congealing cold and blood stasis based on dynamic urine metabonomics and a research method thereof are disclosed, wherein an ultra-high performance liquid chromatography-tandem mass spectrometry technology is adopted to detect and analyze endogenous metabolites which are changed at different time points and used for the cold congealing blood stasis syndrome formed by an ice water bath and adrenalin induced female rat and the prognosis of Chinese angelica root Sini decoction to obtain a fingerprint.
By using a multivariate statistical analysis method, 21 remarkably changed metabolites are screened and identified from multiple variables as differential metabolites potentially related to the development of the congealing cold blood stasis syndrome.
Through a metaboanalyst open source online metabonomics analysis website, 8 metabolic pathways related to different development stages of the blood stasis syndrome due to congealing cold are enriched.
The research preliminarily discusses the diagnostic value of the differential metabolites on the cold coagulation and blood stasis syndrome. Through characteristic curve (ROC) analysis of a subject, the area under the curve (AUC) of 10 metabolites is >0.9, and the area under the curve has higher diagnostic value as a diagnostic index of the cold coagulation blood stasis syndrome.
The specific steps for obtaining the differential metabolite metabolic pathway of the angelica sinensis Sini decoction for resisting cold, blood coagulation and blood stasis are as follows:
(1) preparation of Angelica sinensis Sini decoction
Placing 12g of angelica sinensis, 9g of cassia twig, 9g of white paeony root, 3g of asarum, 6g of ricepaper pith, 6g of honey-fried licorice root and 12g of Chinese date in a 1000mL round-bottom flask, firstly soaking the components in distilled water with the weight being 8 times of the weight of the components for 1h, then carrying out heating reflux extraction at the normal pressure and the temperature of 100 ℃ for 2h, collecting a first extracting solution, and keeping filter residues;
adding water with the weight 6 times of that of the components into a 1000mL round-bottom flask, continuously heating and refluxing the filter residue for extraction for 2 hours, and collecting a second extracting solution;
mixing the first extractive solution and the second extractive solution, and filtering with 200 mesh gauze;
placing the filtered filtrate in a vacuum rotary evaporator, and concentrating at 60 deg.C until the concentration of the final concentrated solution is 2.0g/mL to obtain radix Angelicae sinensis Sini decoction;
(2) determination of animal models
In order to research the influence of the cold coagulation and blood stasis syndrome on the metabolism of rats, a blank control group and a cold coagulation and blood stasis syndrome model group, namely a blank group and a model group, are arranged in the research. Meanwhile, in order to research the intervention effect of the angelica sinensis Sini decoction on the cold coagulation and blood stasis syndrome, the angelica sinensis Sini decoction group, namely the DSD treatment group, is arranged for the rat with the cold coagulation and blood stasis syndrome by gavage in the research;
after 24 female rats are adaptively fed for one week, randomly dividing the rats into a blank group, a model group and a DSD treatment group according to a random digital method, wherein each group comprises 8 rats, the rats in the model group and the DSD treatment group are placed in a cold water bath at 0-2 ℃ for swimming, and when the rats have slow response, weak breathing and stiff legs and are drowned, the rats are taken out and stimulated once a day for 14 days; according to the parallel control principle, the rats in the blank group are placed in a water bath at 35-37 ℃ for swimming for 5-10min, and swim for 1 time every day for 14 days continuously; when the experiment was performed by day 10, rats in the model group and the DSD treatment group were injected subcutaneously with epinephrine hydrochloride twice at 4 hour intervals at a dose of 0.8 mg/kg; 2h after the first injection, the rats in the model group and the DSD treatment group are placed in a cold water bath at 0-2 ℃ for swimming for 5min, and the rats in the blank group are placed in a water bath at 35-37 ℃ for swimming for 5 min; the dosing method of this study was: during the modeling period, the DSD treatment group performs intragastric administration on the angelica sinensis Sini decoction every day with the dose of 1.8mg/g, and the blank group and the model group perform intragastric administration on distilled water with equal dose in parallel once a day for 14 days;
(3) collection, preservation and pretreatment of rat urine
Collecting a required urine sample: on days 0, 5, 10, 14 during animal molding, rats were collected at night 20: 00 to morning 8: 00 urine samples for this period. And urine samples were collected with 5mL centrifuge tubes, respectively. The centrifuge tubes were embedded in ice boxes and each urine sample was at low temperature (0-4 ℃).
Pretreatment and preservation of urine samples: an equal volume of 600. mu.L of urine sample was taken from each sample, and sodium azide as a preservative was added to each urine sample at a mass concentration of 0.1%. The tubes were frozen in a-80 ℃ freezer.
Urine was pre-treated before LC-MS analysis: each urine sample was thawed at room temperature and diluted with an equal volume (600 μ Ι _ of deionized water). Each urine sample was then centrifuged to remove sediment, first at low speed (3500 rpm, 10 min) and then at high speed (12000 rpm, 10 min). And filtering each urine sample through a 0.22 micron polyvinylidene fluoride filter membrane, and filling into a sample injection vial. From each urine sample 10 μ L of urine sample was mixed in 1 new centrifuge tube to make Quality Control (QC) samples for testing the robustness and reproducibility of the method in subsequent liquid chromatography-mass spectrometry analysis.
(4) LC-MS analysis of urine samples
Carrying out method investigation on various groups of rat urine samples collected at different times, establishing an ultra-high performance liquid chromatography-tandem mass spectrometry technology detection analysis rat dynamic urine sample fingerprint analysis method, and carrying out ultra-high performance liquid chromatography-mass spectrometry on the urine samples in sequence;
ultra-high performance liquid chromatography analysis conditions: detecting with ultra-high performance liquid (ACQUITY UPLC) system of Waters corporation, HSST3Chromatographic column (specification of 100mm × 2.1mm, stationary phase particle size of 1.8 μm), experimental column temperature set at 25 deg.C, mobile phase A water, B acetonitrile (containing 0.1% by volume of chromatographic formic acid), and mobile phase flow rate of 0.4 mL/min-1The gradient elution procedure was: 1% of B,0-0.5 min; 1-10% of B,0.5-7 min; 10-50% of B,7-15 min; 50-100% B,15-17min, 100% B,17-19 min; 100-1% B,19-20 min. The temperature of the automatic sample introduction chamber is set to be 4 ℃, and the sample introduction amount is 10 mu L;
mass spectrum conditions: the mass number range of the collected mass spectrum is 100-1000Da by adopting a quadrupole-time-of-flight mass spectrometer (Xevo G2-S Q-TOF) of Waters company for detection. All data were collected in electrospray ion (ESI) source positive ion mode, with capillary voltage 4.0kV, foramen voltage 35kV, extraction voltage 4.0 kV; the ion source temperature is 120 ℃, and the desolventizing gas temperature is 350 ℃; taper hole air flow rate of 40 L.h-1Desolventizing flow rate of 800 L.h-1. Data acquisition is carried out in a Centriod mode by adopting a LockSpray of Waters companyTMThe system carries out real-time correction, takes leucine-enkephalin as a mass reference component (M/z556.2771[ M + H ]]+) The flow rate of the reference solution was 20. mu.L/min. The quality control samples are run through the entire test sequence. 8 urine samples were analyzed in sequence, and one QC sample was injected.
The results are shown in FIGS. 1-4, which show the ultra-high performance liquid chromatograms of the model group rats at 0, 5, 10 and 14 days, and it can be seen from the graphs that the chromatographic behavior of metabolites in the urine of the rats changes at different days.
(5) Data pre-processing
Performing denoising, mass spectrum peak extraction, peak arrangement, peak alignment, normalization and other processing on the acquired ultra performance liquid chromatography and mass spectrum detection data by adopting Masslynx software of Waters company to obtain the peak height or peak area of each peak, retention time and other data; the main processing parameters are as follows: the retention time is 0-20 min; the mass number range is 100-1000 Da; mass number tolerance 0.01; mass number window 0.05; the noise removal level is 6. After the data was subjected to the 80% filtering principle, the variables with blank values > 80% were removed. The normalization method adopts a total ion intensity normalization method.
(6) Metabolic profile differences and dynamics analysis using multivariate variables statistics
The preprocessed data are imported into SIMCA-P14.1 software. First, principal component analysis was performed on the blank group, model group, and QC samples. Before analysis, the data were pre-processed using the Par scaling method. The principal component analysis is used as an unsupervised learning method, and can truly reflect the clustering condition of the samples. As shown in fig. 5, the model group and the blank group are well separated, and have no intersection or overlap, which indicates that there is a significant difference between the two groups of metabolic patterns, and the QC samples are gathered more closely, which indicates that the instrument status is good during the data acquisition process, and the reliability of the data is ensured.
In order to perform dynamic analysis of urine metabolic profile under diseases and preliminary exploration of DSD curative effect, PCA locus analysis is performed on blank group, model group and DSD treatment group urine samples at different time points (day 0, day 5, day 10 and day 14). As shown in fig. 6, the metabolic profiles at day 0 in the PCA tracings for the blank, model and DSD treatment groups were overlapping, indicating that there was no significant difference in the metabolic profiles of the groups at the start of the experiment. As the disease developed, the metabolic profiles of the blank and model groups at days 5, 10 and 14 were gradually separated from each other in the contour map, indicating that the disease had a significant effect on the metabolic profile of rat urine. In this process, the intervention effect of DSD on model group rats was also gradually significant: from day 0 to day 10, DSD had not significantly affected the metabolic profile of BSS rats, but when DSD intervention continued through day 14, the DSD treatment group was significantly differentiated from the metabolic profile of model group rats, indicating that DSD had significant intervention on BSS, resulting in a change in the metabolic profile of model group rats. Principal component analysis each point in the PCA score plot represents the urine metabolism profile of each experimental rat.
In order to further confirm the intervention effect of DSD on BSS rats, partial least squares discriminant analysis was performed on the blank group, the model group and the DSD treatment group, as shown in FIG. 7, the established PLS-DA model fitting ability indexes are R at day 52X＝0.568,R2Y＝0.907,Q20.73; day 10, R2X＝0.588,R2Y＝0.984,Q20.96; day 14, R2X＝0.522,R2Y＝0.96,Q2＝0.751；R2X and R2Y represents the percentage of the PLS-DA classification model that can interpret the X and Y matrix information, Q, respectively2Then, it is calculated by cross-validation to evaluate the prediction power of the PLS-DA model, Q2The larger the model is, the better the prediction effect is; the results show that at the time points of 5, 10 and 14 days, the blank group and the model group are well separated, and the DSD treatment group is between the blank group and the model group, which prompts that the urine metabolic pattern of BSS rats is changed after DSD intervention, and shows that DSD has certain intervention effect on urine metabolic disorder of BSS rats. Each point in the PLS-DA score plot represents the urine metabolism profile of each experimental rat.
(7) Screening for potential biomarkers
As shown in fig. 8, in order to screen differential metabolites that significantly change during the development of BSS, an orthogonal partial least squares analysis (OPLS-DA) model was established using the detection data of ultra performance liquid chromatography and mass spectrometry pretreated by the blank group and the model group, and the differential metabolites between the two groups were screened. Statistical processing was performed using SPSS16.0 software. The screening conditions for differential metabolites were: a Variable Importance Projection (VIP) value >1 was chosen and the difference was statistically significant between the two groups (T-test P value < 0.05). Variables that meet the above conditions will serve as potential biomarkers for BSS. The importance of the variable to the classification was weighed by the size of the VIP value by which the variable was screened in this study.
(8) Identification of potential biomarkers
Identifying potential biomarkers by searching public databases KEGG, HMDB and METLIN according to the precise mass numbers and secondary characteristic fragments mapped by the variables, and comparing the identified potential biomarkers with documents describing metabolite identification mass spectrum information to finally identify twenty differential metabolites related to the development of BSS: 1-methylnicotinamide, creatinine, N-methylhistidine, tyramine glucuronic acid, 1-methylguanine, 1-methyladenine, 5-methylcytosine, 3-hydroxyaminobenzoic acid, leucine-proline, 2-phenylacetamide, indoline, 2-methyl-1, 2,3, 4-tetrahydro-6, 7-isoquinolinediol, xanthurenic acid, glucuronic acid ester of 3-indolecarboxylic acid, hippuric acid, riboflavin, phenylacetylglycine, 5-methoxyindole acetate, corticosterone, allocholic acid, and tetrahydrocorticosterone.
Compared with the blank group, the model group has the content reduction of 8 metabolites, namely 1-methylguanine, 3-hydroxyaminobenzoic acid (3-HAA), indoline, xanthurenic acid, phenylacetylglycine, tetrahydrocorticosterone and 2 unknown metabolites (the m/z values are 182.1181 and 425.2169 respectively) on the 5 th day; at the same time, the content of hippuric acid and 5-methoxyindolacetic acid is increased.
At day 10 of the model group, the number of metabolites showed a significant decrease of 12, specifically: 1-methylnicotinamide, creatinine, N-methylhistidine, 1-methylguanine, 1-methyladenine, 5-methylcytosine, 3-HAA, indoline, xanthurenic acid, riboflavin, corticosterone, tetrahydrocorticosterone, two unknown metabolites with m/z values of 182.1181 and 425.2169, respectively). In contrast, on day 10, tyramine glucuronic acid, leucine-proline, 2-phenylacetamide, 2-methyl-1, 2,3, 4-tetrahydro-6, 7-isoquinolinediol, 3-indolecarboxylic acid glucuronic acid and hippuric acid content increased.
The model group had a reduced content of 2-methyl-1, 2,3, 4-tetrahydro-6, 7-isoquinolinediol, 3-indolecarboxylic acid glucuronate, 5-methoxyindole acetate and allocholic acid on day 14. By analyzing the content of the different metabolites in the DSD treatment group, it was found that 4 metabolites can be significantly recalled by DSD with respect to the model group and act at different times, specifically: 5-methoxyindole acetate and indoline were recalled by DSD on day 5; 3-indolecarboxylic acid glucuronic acid was recalled at 10 by DSD; the 2-methyl-1, 2,3, 4-tetrahydro-6, 7-isoquinolinediol was recalled by DSD on days 10 and 14. Urine differential metabolites and content trend of rats in different period blank group, model group and DSD treatment group are shown in table 1.
TABLE 1 urine differential metabolite and content variation trends of rats in different time blank groups, model groups and DSD treatment groups
(M/C represents model versus normal group:#P<0.05,##P<Q/M represents DSD treatment group versus model group:*P<0.05,**P<0.01)
(9) and (3) metabolic pathway enrichment analysis: uploading the screened and identified differential metabolites to a metabonalyst metabonomics online analysis webpage, inputting urine metabolite names on different days, standardizing the metabolite names, and selecting a KEGG database of rat species for enriching related pathways. As shown in fig. 9, the significantly affected metabolic pathways in urine of BSS rats at different periods are subjected to enrichment analysis and topology analysis, and 8 metabolic pathways are obtained by total enrichment, so that an analysis metabolic pathway map is constructed based on the metabolic pathways; the influence value obtained by metabolic pathway analysis, namely the Impact value, when the Impact is greater than 0.1, the metabolic pathway has important influence in a metabolic network. Therefore, the determination of the interconversion of nicotinic acid and nicotinamide metabolism (Impact value of 0.125) and pentose and glucuronic acid conversion (Impact value of 0.273) according to the pathway importance values of the metabolic pathways is the two most relevant metabolic pathways for the development of BSS. As shown in FIG. 10, the pathways involved in metabolites were summarized, and a metabolic pathway summary map was drawn. The relationship between pathways and metabolites and the dynamic effects between pathways can be seen in the figure.
① dynamic analysis of urine could reveal the whole process of disease development and drug regulation, and help the intensive study of disease and treatment mechanism at 5, 10 and 14 days, comparing the content difference of metabolites between model group and blank group, finally 21 metabolites were identified as biomarkers.
② Tryptophan plays an essential role in mammalian protein biosynthesis, which is metabolized primarily by two pathways, firstly, the main pathway of tryptophan catabolism is kynurenine metabolism, which is a key step in the production of a number of important biologically active metabolites (e.g., antagonist kynurenine, neuroactive kynurenine, zinc binding compound picolinic acid). This metabolite xanthurenic acid, which is a substrate for methyltransferase, is a metabolite with photochemical activity.Metabolic product 3-HAA, which is a key metabolic intermediate after tryptophan has undergone a series of enzymatic reactions.in our study, model rats have reduced levels of xanthurenic acid and 3-HAA on days 5 and 10 compared to blank rats.reduction of 3-HAA directly affects the conversion of 3-HAA to hydroxyquinolinone, while hydroxyquinolinone is a key metabolic intermediate linking tryptophan metabolism with nicotinic acid and nicotinamide, secondly, which tryptophan is an essential precursor for the synthesis of tryptophane, a neurotransmitter, which plays an important role in mood, stress response, and the downstream of tryptophan metabolism, which is a potential for the regulation of indole function on days 5-5, which is significantly impaired by serum acetic acid, which is a metabolite that is involved in the serum contraction that is a serum metabolite that is likely to be increased on days 5-14, but that is likely to be induced by a decrease in the normal serum contraction of indole metabolism.
③ Niacin and Niacin metabolism are important metabolic pathways in organisms Niacin is an amidated form of Niacin, has antioxidant stress and anti-inflammatory effects, is involved in cellular energy metabolism, effectively protects cell membranes from free radical damage, prevents activation of inflammatory cells Niacin is converted to 1-methylnicotinamide under the action of Niacin N-methyltransferase (EC:2.1.1.1) and metabolized to 1-methyl-4-pyridine-5-carboxamide under the action of aldehyde oxidase (EC: 1.2.3.1.) the results show that 1-methylnicotinamide is significantly reduced on day 10 in the model group compared to the blank group, which may lead to abnormal Niacin and Niacin metabolism in the pathological state of BSS.
④ glycolysis or gluconeogenesis plays a crucial role in human body, providing a source of glucose molecules, glucuronic acid is closely related to glucose metabolism in vivo under the action of glucuronic acid transferase (EC:2.4.1.17), glucuronic acid can be converted into glucose aldoside with strong hydrophilicity, and is more easily excreted by the body.
⑤ phenylalanine is synthesized as an amino acid into various cellular proteins, which are mainly converted into tyrosine for the synthesis of certain hormones and neurotransmitters, such as dopamine, epinephrine and norepinephrine, therefore, stable phenylalanine metabolism is a key factor for maintaining normal growth and physiological functions.
⑥ the general process of steroid production includes a pathway from cholesterol to progesterone the acute regulatory protein of steroidogenesis (StAR) is a protein that binds cholesterol and promotes the efflux of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, arachidonic acid is essential for steroid formation and StAR expression in cells, steroid synthesis is affected when arachidonic acid synthesis is blocked, previous studies have shown that serum arachidonic acid and the corticosterone precursor 21-deoxycorticosterol are reduced when BSS occurs, leading to a reduction in metabolites associated with the steroid synthesis pathway in this study, corticosterone is reduced on day 10 of the model group compared to the blank group, BSS is also significantly reduced on days 5, 10 and 14.
(10) Receiver characteristic curve (ROC) analysis: the original data of the 21 identified metabolites are introduced into SPSS16.0 software for ROC analysis, and a series of areas under ROC curves (AUC) are obtained, wherein AUC of 10 metabolites is greater than 0.9, which indicates that the 10 metabolites have higher diagnostic value as diagnostic indicators of blood stasis due to cold coagulation. The ROC results for each metabolite are shown in table 2.
TABLE 2 ROC results for each metabolite
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (6)
1. The angelica sinensis Sini decoction is used for resisting cold, coagulating blood stasis and different metabolite metabolic pathways and a research method, and is characterized by comprising the following steps:
adopting ultra performance liquid chromatography tandem mass spectrometry to detect and analyze endogenous metabolites which are induced by an ice water bath and adrenalin to form congealing cold blood stasis syndrome and angelica sinensis Sini soup stem prognosis and change at different time points to obtain a fingerprint;
utilizing a multivariate variable statistical analysis method to screen and identify twenty-one metabolites with significant changes from a plurality of variables as differential metabolites potentially related to the occurrence and development of congealing cold blood stasis;
eight metabolic pathways related to different development stages of the blood stasis syndrome due to congealing cold are enriched through a metaboanalyst open source online metabonomics analysis website.
2. The angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and the research method thereof as claimed in claim 1, wherein the specific steps for obtaining the angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway are as follows:
(1) preparation of Angelica sinensis Sini decoction
Placing 12g of angelica sinensis, 9g of cassia twig, 9g of white paeony root, 3g of asarum, 6g of ricepaper pith, 6g of honey-fried licorice root and 12g of Chinese date in a 1000mL round-bottom flask, firstly soaking the components in distilled water with the weight being 8 times of the weight of the components for 1h, then carrying out heating reflux extraction at the normal pressure and the temperature of 100 ℃ for 2h, collecting a first extracting solution, and keeping filter residues;
adding water with the weight 6 times of that of the components into a 1000mL round-bottom flask, continuously heating and refluxing the filter residue for extraction for 2 hours, and collecting a second extracting solution;
mixing the first extractive solution and the second extractive solution, and filtering with 200 mesh gauze;
placing the filtered filtrate in a vacuum rotary evaporator, and concentrating at 60 deg.C until the concentration of the final concentrated solution is 2.0g/mL to obtain radix Angelicae sinensis Sini decoction;
(2) determination of animal models
After 24 female rats are adaptively fed for one week, randomly dividing the rats into a blank group, a model group and a DSD treatment group according to a random digital method, wherein each group comprises 8 rats, the rats in the model group and the DSD treatment group are placed in a cold water bath at 0-2 ℃ for swimming, and when the rats have slow response, weak breathing and stiff legs and are drowned, the rats are taken out and stimulated once a day for 14 days; according to the parallel control principle, the rats in the blank group are placed in a water bath at 35-37 ℃ for swimming for 5-10min, and swim for 1 time every day for 14 days continuously; when the experiment was performed by day 10, rats in the model group and the DSD treatment group were injected subcutaneously with epinephrine hydrochloride twice at 4 hour intervals at a dose of 0.8 mg/kg; 2h after the first injection, the rats in the model group and the DSD treatment group are placed in a cold water bath at 0-2 ℃ for swimming for 5min, and the rats in the blank group are placed in a water bath at 35-37 ℃ for swimming for 5 min;
the dosing method of this study was: during the modeling period, the DSD treatment group performs intragastric administration on the angelica sinensis Sini decoction every day with the dose of 1.8mg/g, and the blank group and the model group perform intragastric administration on distilled water with equal dose in parallel once a day for 14 days;
(3) collection, preservation and pretreatment of rat urine
On days 0, 5, 10, 14 during animal molding, rats per group were collected at night 20: 00 to morning 8: 00, collecting urine samples in the time period by using 5mL centrifuge tubes respectively, and burying each centrifuge tube in an ice box to enable each urine sample to be at 0-4 ℃;
after an equivalent 600 mu L urine sample is taken out from each urine sample, adding 0.1 mass percent sodium azide into each urine sample, and then freezing and storing each centrifugal tube at the temperature of minus 80 ℃;
unfreezing each urine sample at room temperature, diluting each urine sample with 600 mu L of deionized water, centrifuging each urine sample to remove precipitates, filtering each urine sample through a 0.22 micron polyvinylidene fluoride filter membrane, respectively filling the urine samples into sample injection vials, and then taking 10 mu L of urine sample from each sample and mixing the urine sample in 1 new centrifugal tube to prepare a quality control QC sample;
(4) ultra-high performance liquid chromatography tandem mass spectrometry detection analysis of urine sample
(5) Data pre-processing
Performing denoising, mass spectrum peak extraction, peak arrangement, peak alignment and normalization processing on the acquired ultra performance liquid chromatography and mass spectrum detection data by adopting Masslynx software to obtain the peak height or peak area and retention time of each peak;
(6) metabolic profile differences and dynamics analysis using multivariate variables statistics
Importing the preprocessed data into SIMCA-P14.1 software, preprocessing the data by adopting a Par scaling method, then carrying out principal component analysis on blank groups, model groups and QC samples, carrying out PCA (principal component analysis) track analysis on the blank groups, the model groups and the DSD (differential motion detection) treatment group urine samples collected at different time points, and carrying out partial least square discriminant analysis on the blank groups, the model groups and the DSD treatment groups;
(7) screening for potential biomarkers
Establishing an orthogonal partial least square analysis model by using the detection data of the ultra performance liquid chromatography and the mass spectrum after the pretreatment of the blank group and the model group, screening differential metabolites between the two groups, and performing statistical processing by adopting SPSS16.0 software; the screening conditions for differential metabolites were: selecting a projection value of variable importance >1, wherein the difference has statistical significance between the two groups;
(8) identification of potential biomarkers
And (3) identifying potential biomarkers by searching public databases KEGG, HMDB and METLIN according to the accurate mass number and the secondary characteristic fragments corresponding to the variables, and comparing the potential biomarkers with a document which describes the identification mass spectrum information of the metabolites to finally identify twenty differential metabolites related to the occurrence and development of BSS: 1-methylnicotinamide, creatinine, N-methylhistidine, tyramine glucuronic acid, 1-methylguanine, 1-methyladenine, 5-methylcytosine, 3-hydroxyaminobenzoic acid, leucine-proline, 2-phenylacetamide, indoline, 2-methyl-1, 2,3, 4-tetrahydro-6, 7-isoquinolinediol, xanthurenic acid, glucuronic acid ester of 3-indolecarboxylic acid, hippuric acid, riboflavin, phenylacetylglycine, 5-methoxyindole acetate, corticosterone, allocholic acid, tetrahydrocorticosterone;
(9) metabolic pathway enrichment assay
Uploading the identified twenty-one differential metabolite to a metaboanalyst open source online metabonomics analysis website, inputting urine metabolite names of different days, standardizing the metabolite names, and selecting a KEGG database of rat species to enrich related pathways; carrying out enrichment analysis and topological analysis on the significantly influenced metabolic pathways in urine of BSS rats in different periods, and obtaining eight metabolic pathways by total enrichment: nicotinic acid and nicotinamide metabolic pathway, starch and sucrose metabolic pathway, histidine metabolic pathway, pentose and glucuronic acid metabolic pathway, tryptophan metabolic pathway, phenylalanine metabolic pathway, riboflavin metabolic pathway, and steroid hormone biosynthetic metabolic pathway.
3. The angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and the research method of the angelica sinensis Sini decoction as claimed in claim 2, wherein the ultra performance liquid chromatography detection adopts an ultra performance liquid system of Waters company for detection, and HSST (HSST)3A chromatographic column with specification of 100mm multiplied by 2.1mm, fixed phase particle size of 1.8 μm, experimental column temperature of 25 ℃, mobile phase A water, B acetonitrile containing chromatographic formic acid, wherein, the volume fraction of the chromatographic formic acid is 0.1%, and the flow rate of the mobile phase is 0.4 mL/min-1The gradient elution procedure was: 1% of B,0-0.5 min; 1-10% of B,0.5-7 min; 10-50% of B,7-15 min; 50-100% B,15-17min, 100% B,17-19 min; 100-1% B,19-20 min; the temperature of the automatic sample introduction chamber was set to 4 ℃ and the sample introduction amount was 10. mu.L.
4. The angelica Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and the research method as claimed in claim 2, wherein the mass spectrometry detection adopts a quadrupole-time-of-flight mass spectrometer of Waters company for detection, the mass number range of the collected mass spectrum is 100-1000Da, all data are collected in an electrospray ion ESI source positive ion mode,wherein the capillary tube voltage is 4.0kV, the vertebral foramen voltage is 35kV, and the extraction voltage is 4.0 kV; the ion source temperature is 120 ℃, and the desolventizing gas temperature is 350 ℃; taper hole air flow rate of 40 L.h-1Desolventizing flow rate of 800 L.h-1Data acquisition in the Centriod model, using LockSpray from WatersTMThe system carries out real-time correction, leucine-enkephalin is taken as a mass reference component, the flow rate of a reference solution is 20 mu L/min, 8 urine samples are analyzed in the sequence, and a QC sample is injected once.
5. The angelica sinensis Sini decoction cold-resistant blood-coagulation-stasis-syndrome-resisting differential metabolite metabolic pathway and the research method of claim 2, wherein main processing parameters of data preprocessing are as follows: the retention time is 0-20 min; the mass number range is 100-1000 Da; mass number tolerance 0.01; mass number window 0.05; noise removal degree 6, after data was processed through 80% filtering principle, the variable with blank value > 80% was removed.
6. The angelica sinensis Sini-decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and the research method as claimed in claim 2, wherein when each urine sample is centrifuged to remove precipitates, the urine sample is firstly centrifuged at 3500 rpm for 10 minutes at a low speed, and then centrifuged at 12000 rpm for 10 minutes at a high speed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911128114.3A CN110794074A (en) | 2019-11-18 | 2019-11-18 | Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911128114.3A CN110794074A (en) | 2019-11-18 | 2019-11-18 | Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110794074A true CN110794074A (en) | 2020-02-14 |
Family
ID=69445079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911128114.3A Pending CN110794074A (en) | 2019-11-18 | 2019-11-18 | Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110794074A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647640A (en) * | 2020-05-22 | 2020-09-11 | 中国药科大学 | Method for rapidly and accurately realizing classification of cardiac function and course of chronic heart failure |
| CN113049664A (en) * | 2021-03-15 | 2021-06-29 | 东华理工大学 | Path analysis modeling method based on mass spectrum metabonomics |
| CN113984935A (en) * | 2021-11-17 | 2022-01-28 | 东莞理工学院 | Method for researching metabolic characteristics of acetoacidophilic proteophilus based on metabolome analysis |
| CN114414671A (en) * | 2021-12-10 | 2022-04-29 | 天津中医药大学 | Research method for metabolic pathway of diabetes differential metabolites by using coreopsis bicolor |
| CN115290797A (en) * | 2022-09-13 | 2022-11-04 | 贵州医科大学 | Metabonomics research method for anti-rheumatoid arthritis of periploca forrestii based on gas chromatography-mass spectrometry technology |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361953A (en) * | 2008-09-22 | 2009-02-11 | 山西中医学院 | Use of Sini decoction in preparing medicine for treating hypothyroidism sexual function reduction |
| CN103989163A (en) * | 2014-04-30 | 2014-08-20 | 新时代健康产业（集团）有限公司 | Blood stasis physique health food and making method thereof |
| CN104524545A (en) * | 2014-12-11 | 2015-04-22 | 南京中医药大学 | Model making method for cold-coagulation blood-stasis type primary dysmenorrheal animal model |
| CN108414635A (en) * | 2018-02-13 | 2018-08-17 | 广西医科大学 | The difference metabolin metabolic pathway and research method of a kind of anti-grease toxicity of rubusoside based on cell metabolism group |
| CN109115933A (en) * | 2018-08-31 | 2019-01-01 | 中国人民解放军第四军医大学 | A method of screening Protective effects of Sini decoction myocardial ischemia effective substance |
-
2019
- 2019-11-18 CN CN201911128114.3A patent/CN110794074A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361953A (en) * | 2008-09-22 | 2009-02-11 | 山西中医学院 | Use of Sini decoction in preparing medicine for treating hypothyroidism sexual function reduction |
| CN103989163A (en) * | 2014-04-30 | 2014-08-20 | 新时代健康产业（集团）有限公司 | Blood stasis physique health food and making method thereof |
| CN104524545A (en) * | 2014-12-11 | 2015-04-22 | 南京中医药大学 | Model making method for cold-coagulation blood-stasis type primary dysmenorrheal animal model |
| CN108414635A (en) * | 2018-02-13 | 2018-08-17 | 广西医科大学 | The difference metabolin metabolic pathway and research method of a kind of anti-grease toxicity of rubusoside based on cell metabolism group |
| CN109115933A (en) * | 2018-08-31 | 2019-01-01 | 中国人民解放军第四军医大学 | A method of screening Protective effects of Sini decoction myocardial ischemia effective substance |
Non-Patent Citations (1)
| Title |
|---|
| HUIMIN HUANG等: "Dynamic urinary metabolomics analysis based on UHPLC-Q-TOF/MS to investigate the potential biomarkers of blood stasis syndrome andthe effects of Danggui Sini decoction", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647640A (en) * | 2020-05-22 | 2020-09-11 | 中国药科大学 | Method for rapidly and accurately realizing classification of cardiac function and course of chronic heart failure |
| CN113049664A (en) * | 2021-03-15 | 2021-06-29 | 东华理工大学 | Path analysis modeling method based on mass spectrum metabonomics |
| CN113984935A (en) * | 2021-11-17 | 2022-01-28 | 东莞理工学院 | Method for researching metabolic characteristics of acetoacidophilic proteophilus based on metabolome analysis |
| CN113984935B (en) * | 2021-11-17 | 2024-04-02 | 东莞理工学院 | Method for researching metabolic characteristics of acetoacidophile based on metabonomic analysis |
| CN114414671A (en) * | 2021-12-10 | 2022-04-29 | 天津中医药大学 | Research method for metabolic pathway of diabetes differential metabolites by using coreopsis bicolor |
| CN115290797A (en) * | 2022-09-13 | 2022-11-04 | 贵州医科大学 | Metabonomics research method for anti-rheumatoid arthritis of periploca forrestii based on gas chromatography-mass spectrometry technology |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110794074A (en) | Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method | |
| CN108508055B (en) | Metabonomics-based diabetes resistance potential marker metabolic pathway of Guangxi Yaoshan sweet tea and research method | |
| Kumari et al. | Quantitative metabolomics of saliva using proton NMR spectroscopy in patients with Parkinson’s disease and healthy controls | |
| CN107247095B (en) | A kind of construction method of the rat model for oxidation-resisting and caducity drug screening based on metabonomic analysis | |
| Barderas et al. | Metabolomic profiling for identification of novel potential biomarkers in cardiovascular diseases | |
| CN110646554A (en) | Pancreatic cancer diagnosis marker based on metabonomics and screening method and application thereof | |
| EP2685259A1 (en) | A method of diagnosing an asthma disease state | |
| CN107389842B (en) | Xinshao prescription anti-cerebral ischemia reperfusion injury metabonomic research method based on GC-MS combined technology | |
| Zhang et al. | Lipidomics to investigate the pharmacologic mechanisms of ginkgo folium in the hyperuricemic rat model | |
| CN109239224B (en) | Method for simultaneously and quantitatively measuring 9 blood-entering components in spina date seed water extract | |
| CN106526156A (en) | Method for detecting, screening and identifying kidney-yang deficiency metabolism biomarkers | |
| CN104713970B (en) | A kind of construction method of blood serum metabolic group analysis model | |
| CN109507337A (en) | A kind of new method based on blood urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism | |
| Pietrowska et al. | LC-MS-based metabolic fingerprinting of aqueous humor | |
| Zhao et al. | Exploring potential biomarkers and determining the metabolic mechanism of type 2 diabetes mellitus using liquid chromatography coupled to high-resolution mass spectrometry | |
| CN108334752A (en) | Establish the method and healthy human urine's proteome databases of healthy human urine's protein group qualitative reference range | |
| Liu et al. | Metabolomic study of a rat fever model induced with 2, 4-dinitrophenol and the therapeutic effects of a crude drug derived from Coptis chinensis | |
| Gao et al. | A urinary metabonomics study on biochemical changes in yeast-induced pyrexia rats: A new approach to elucidating the biochemical basis of the febrile response | |
| CN109142419A (en) | A kind of potential marker metabolic pathway of serum and research method of the moringa seeds anti-diabetic based on metabolism group | |
| CN106153739A (en) | A kind of model based on metabonomic technology diagnosing liver cancer | |
| Li et al. | Analysis of therapeutic effect of Ilex hainanensis Merr. extract on nonalcoholic fatty liver disease through urine metabolite profiling by ultraperformance liquid chromatography/quadrupole time of flight mass spectrometry | |
| CN110879297A (en) | Lipid biomarker for detecting nephrotic syndrome rat model and application thereof | |
| CN116413432A (en) | Pancreatic cancer diagnosis marker based on intestinal flora metabonomics, screening method and application thereof | |
| CN116482380A (en) | Use of biomarkers for depression | |
| CN114414671A (en) | Research method for metabolic pathway of diabetes differential metabolites by using coreopsis bicolor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200214 |