CN111896728A - Combined marker for detecting anti-depression amount-effect and amount-toxicity relationship of bupleurum petroleum ether part and application thereof - Google Patents
Combined marker for detecting anti-depression amount-effect and amount-toxicity relationship of bupleurum petroleum ether part and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of application of endogenous micromolecular metabolites of biological samples, and provides a combined marker for detecting the relation between antidepressant quantity-effect and quantity-toxicity of bupleurum petroleum ether parts and application thereof, wherein the quantity-effect marker comprises the following components: l-isoleucine/leucine, L-carnitine, L-acetyl-carnitine, adenine, 2-phenylacetamide; the quantity-toxicity markers include: l-carnitine, valine, malic acid, 2' -adenosine phosphate, and 2-ascorbic acid sulfate. The invention is used for evaluating the relationship between the antidepressant dose-effect and the dose-toxin of the bupleurum petroleum ether part and solves the problem of dose application of the traditional Chinese medicine bupleurum in the evaluation of the efficacy and the safety of treating the depression. Sensitively and intuitively evaluate the relationship between the in-vivo action and the dosage of the petroleum ether part of the bupleurum, provide technical support for the efficacy and the toxic dosage of the traditional Chinese medicine bupleurum, and provide a new method for researching the effectiveness and the safety of the traditional Chinese medicine bupleurum.
Description
Technical Field
The invention belongs to the technical field of application of endogenous micromolecular metabolites of biological samples, and particularly relates to a combined marker for detecting the relation between antidepressant amount-effect and amount-toxicity of bupleurum petroleum ether parts and application thereof. Detecting endogenous micromolecule compounds in a biological sample by adopting a liquid phase-mass spectrum coupling technology, screening the compounds by adopting a multivariate data processing method, and obtaining a dose-effect and dose-toxicity index by combining a mathematical model for evaluating a dose-drug/toxicity relation.
Background
The dose-effect/toxicity relationship of traditional Chinese medicine is the essence of clinical medication of traditional Chinese medicine, and for a long time, due to the action modes of multiple components and multiple targets of the traditional Chinese medicine, how to comprehensively evaluate the dose-effect/toxicity relationship in an integral angle is a major subject which needs to be solved in the modernization process of the traditional Chinese medicine.
However, although the existing evaluation mode in the quantity-effect/toxicity research adopts some convenient and effective evaluation indexes, such as weight, blood pressure, blood sugar, transaminase, platelets, cell number and the like, the effect indexes of many diseases are lack of quantification, and the existing method is difficult to implement accurate detection and evaluation. Such as the degree of depression and anxiety, the degree of fatigue, the degree of pain and analgesic effect, the strength of rejection reactions of the body and the effect of immunosuppressive agents, etc.
Meanwhile, the existing evaluation standards lack objective quantitative indexes, and subjective artificial evaluation or qualitative evaluation is adopted in the drug effect evaluation process, such as tissue slice, imaging technology, various electron microscope examinations and the like. In addition, although the metabonomics technology is adopted as the drug effect evaluation, the metabonomics data volume is huge, the evaluation mode lacking focusing is difficult to popularize and apply in clinic, in a word, the existing quantity-effect toxicity evaluation method has large limitation, lacks quantitative standard, lacks objectivity, comprehensiveness, pertinence and innovativeness, is not beneficial to objectively and accurately evaluating the traditional Chinese medicine quantity-effect/toxicity, and becomes a huge obstacle severely restricting pharmacological research and troubles the pharmacological research. Research and establishment of a quantitative-effective toxicity evaluation method which is convenient, reasonable and widely applied are urgently needed.
Disclosure of Invention
The invention provides a combined marker for detecting the relation between the antidepressant amount-effect and the amount-toxicity of bupleurum petroleum ether parts and application thereof, which comprehensively and quantitatively detects endogenous micromolecules in biological samples, focuses on a differential metabolite group capable of reflecting the relation between the amount-effect and the toxicity, integrates a mathematical model and is finally applied to the evaluation of the relation between the amount and the effect.
The invention is realized by the following technical scheme: a combined marker for detecting the relation between antidepressant amount-effect and amount-toxicity of bupleurum petroleum ether parts, wherein the amount-effect marker comprises: l-isoleucine/leucine, L-carnitine, L-acetyl-carnitine, adenine, 2-phenylacetamide; the quantity-toxicity markers include: l-carnitine, valine, malic acid, 2' -adenosine phosphate, and 2-ascorbic acid sulfate.
The screening method of the combined marker comprises the following steps: detecting the metabolite change in the liver sample before and after normal rats and CUMS model rats are given bupleurum petroleum ether parts by using a high-resolution mass spectrometry technology, screening differential metabolites, selecting quantity-effect and quantity-toxicity metabolites to obtain a quantity-effect and quantity-toxicity marker combination, and screening out the combined marker based on the calculation of the quantity-effect and quantity-toxicity indexes of a mathematical model.
The specific screening method comprises the following steps:
(1) unpredictable stress rats, namely CUMS model rats and normal rats are treated by the drug at the part of the bupleurum petroleum ether, and the relationship between the drug quantity-effect and the drug quantity-toxicity is determined according to the behavioral test and the biochemical index test;
(2) respectively detecting metabolite analysis in the biological samples before and after the drug treatment by using an analysis method of a metabonomics determination technology; carrying out metabolic profile analysis on the obtained analysis map to obtain a data set; screening potential biomarkers with antidepressant effect and toxicity by using a multivariate variable statistical analysis method for the data set; and (2) screening the quantity-effect metabolic markers and quantity-toxicity markers of the antidepressant effect and toxicity on the content change of the biomarkers in biological samples of the CUMS model rat and the normal rat by using the bupleurum petroleum ether part with the medicine effective dose gradient determined in the step (1).
The biological sample is: liver, whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, sweat, tissue, cells, or cell culture fluid.
In the step (1): in the medical treatment of the bupleurum petroleum ether part, the medicine dose is as follows: the crude drugs are respectively 1, 3, 6, 12.5, 25, 50 and 100 g/kg; the administration dose of each group of rats is 10 mL/kg/d; the behavioral test was: weighing, open field experiments and sweet water preference experiments; the biochemical index test is as follows: alanine aminotransferase ALT, aspartate aminotransferase AST, alkaline phosphatase ALP, total bilirubin TBIL, urea nitrogen BUN and creatinine CREA were measured with a full-automatic biochemical analyzer, respectively.
The metabonomics determination technology in the step (2) is liquid chromatogram-high resolution mass spectrum UHPLC-Q-Orbitrap-MS, a mass spectrum determination technology or a nuclear magnetic resonance determination technology.
In the step (2), UHPLC-QOxacttive orbit-MS is adopted for determination, and the specific method comprises the following steps: chromatographic conditions are as follows: liquid phase conditions: mobile phase: a: water, containing 0.1% formic acid, B: acetonitrile, containing 0.1% formic acid; gradient of mobile phase: 0-2 min, 2% B; 2-3 min, 2-35% of B; 3-17 min, 35-70% B; 17-18 min, 70% B; 18-29 min, 70-98% B; 29-31 min, 98% B; 31-33 min, 98-2% B; 33-35 min, 2% B. Flow rate of 0.2ml/min, 5. mu.L sample size, column temperature 40 ℃, Waters ACQUITYUPLC HSS T3 column: 2.1 × 100mm, 1.7 μm;
mass spectrum conditions: adopting an ESI electrospray ionization mode; switching an acquisition mode by positive and negative ions, wherein the scanning mode comprises the following steps: FullScan/dd-MS2, wherein the m/z acquisition range is 100-1500; the positive electrode of the spraying voltage is 3.5 kV; the negative electrode is 2.5 kV; the capillary temperature was 320 ℃; the temperature of the heater is 300 ℃; flow rate of sheath gas: 35arb, assist gas flow rate: 10 arb; resolution was set at MS FullScan 35000 FWHM and MS/MS 17500FWHM, NCE was set at 12.5eV, 25eV and 37.5 eV;
the specific test method for the obtained LC-MS spectrum data comprises the following steps: and importing the acquired LC-MS/MS data file into Compound discover 2.0 software to acquire matched and aligned peak data, wherein the set parameters are as follows: mass range (mass range): 100-1500 Da; mass deviation (MassTolerance): 5 ppm; RT tolerance [ min ]: 0.05; signal-to-noise threshold (S/NThreshold): 1.5;
leading the peak data containing the retention time, molecular formula, accurate molecular weight and peak area information into Excel for peak area normalization storage; finally, the data after the peak area normalization is led into SIMCA-P13.0 to carry out partial least square discriminant analysis PLS-DA and orthogonal partial least square discriminant analysis OPLS-DA; simultaneously screening the difference variable with the largest contribution by combining VIP >1 in an S-plot and P <0.05 of t test of an independent sample;
and identifying the screened differential variables, introducing the peak areas and corresponding dosage information into SPSS (SpSS) for carrying out Pearson correlation analysis, and screening out differential metabolites with strong correlation.
The specific dose-effect/toxicity index relation calculation adopts a pharmacodynamic index EI mathematical model, partial least square-discriminant analysis PLS-DA, orthogonal partial least square method OPLS, orthogonal partial least square method-discriminant analysis OPLS-DA, principal component analysis PCA, nonlinear mapping NLM or cluster analysis HCA mathematical model.
The specific dose-effect/toxicity index relation is calculated by adopting a pharmacodynamic index EI mathematical model, and the calculation method comprises the following steps: the calculation formula of the effect value EI and the toxicity value is as follows:
wherein: ki represents the average value of the content of a certain metabolite in the blank group; mi represents the average value of the corresponding metabolite content in the model group; ci represents the content of corresponding metabolites in the model medicine group; zi represents the content of corresponding metabolite in the blank administration group; and carrying out linear regression analysis on the dose effect/toxicity index to obtain a dose effect/toxicity curve.
The application of the combined marker is characterized in that: the combined marker is used for evaluating the relation between the depression resisting amount and the effect/toxicity of the bupleurum.
The invention adopts high resolution mass spectrometry to detect the metabolite change in liver samples before and after normal rats and CUMS model rats are fed with bupleurum petroleum ether parts, obtains the marker combination of quantity-effect and quantity-toxicity by screening different metabolites and selecting quantity-effect and quantity-toxicity metabolites, and is used for evaluating the relation of antidepressant quantity-effect and quantity-toxicity of bupleurum petroleum ether parts based on the calculation of quantity-effect and quantity-toxicity indexes of a mathematical model, thereby solving the problem of dose application of traditional Chinese medicine bupleurum in the evaluation of efficacy and safety of treating depression. Compared with the conventional quantity-effect and quantity-toxicity evaluation method, the method has the advantages that the relation between the in-vivo effect and the dosage of the petroleum ether part of the Chinese thorowax root is evaluated sensitively and visually, the technical support is provided for the efficacy and the toxic dosage of the Chinese thorowax root, and a new method is provided for exploring the effectiveness and the safety of the Chinese thorowax root.
Drawings
FIG. 1 is a UHPLC-Q-Orbitrap-MS measurement of dose-effect (A) and dose-toxicity (B) metabolic profiles;
FIG. 2A is a graph of dose-effect-toxicity differential metabolite properties; FIG. 2B is a dose-effect differential metabolite linear relationship; FIG. 2C is a dose-differential metabolite linear relationship;
FIG. 3A is the dose-effect indices of the groups C4, C6 and C7; FIG. 3C is a C4, C6 and C7 group dose-effect curve, and FIG. 3B is a Z4, Z6 and Z7 group dose-toxicity index; FIG. 3D is a Z4, Z6, and Z7 group toxicity curve.
Detailed Description
The present invention is explained in detail by the following examples, but the present invention is not limited thereto.
1. Protocol and sample collection: 136 male SD rats, randomly divided into 2 groups, i.e., CUMS model group (n =72) and normal group (n = 64). Wherein, the CUMS model group rats are randomly divided into 9 groups (n = 8), namely a model group (CM), a venlafaxine hydrochloride group (CY, 0.035 g/kg) and a bupleurum petroleum ether part 7 dosage administration group (C1-C7, crude drug amount 1, 3, 6, 12.5, 25, 50 and 100 g/kg); the normal group rats were randomly divided into 8 groups (n = 8), i.e., a blank group (K), 7 dose administration groups of bupleurum petroleum ether sites (Z1-Z7, crude drug amount 1, 3, 6, 12.5, 25, 50 and 100 g/kg). The rats of each group of the CUMS model group are singly raised in one single arc, and the rats are subjected to intragastric administration while being modeled, stimulation is given 1h after administration, and the implementation of each stimulation is random and discontinuous and lasts for 21 d; the rats in the normal group are fed with 4 rats per cage, and are simultaneously administered with the CUMS model group by intragastric administration for 21 days, and the rats are normally fed without molding. In addition, blank solvents used for dissolving the drugs in the experiment are 0.5 percent sodium carboxymethylcellulose-0.5 percent tween-80 aqueous solution, the blank solvents with the same dosage are administered to rats in the K group and the CM group by intragastric administration, and the administration dosage of each group of rats is 10 mL/kg/d.
The models were replicated after modification based on the methods reported in the literature for rats in CUMS model groups, wherein the stimulating factors include water deprivation for 24h, heat stimulation at 50 ℃ for 5min, ultrasound for 3h, tail clamping for 2min, restraint for 3h, electric shock (36 v, 10 times), fasting for 24h, reversal of day and night for 12h, and ice water swimming at 4 ℃ for 5 min. The rats in the blank group were kept normally for the rest of the time without any stimulation except that water was not allowed for 24 hours before the sugar water consumption experiment. CUMS model replication consisted of 21-day random challenge modeling, the challenge type of which is shown in table 1.
Table 1: stimulation type table of CUMS rats
In the experimental process, behavioral tests including weight weighing, open field experiments and sugar water preference experiments are carried out at 0d, 7d, 14d and 21d respectively. Each group of rats was subjected to orbital bleeding 1.5ml before administration (0 d), 7d, 14d and 21d, and after standing in a 2ml EP tube for 0.5h, centrifuged at 13000r/min at 4 ℃ for 10min, and the serum was transferred to another EP tube and stored at-80 ℃ for further use.
Testing biochemical indexes of liver and kidney: alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), Total Bilirubin (TBIL), urea nitrogen (BUN) and Creatinine (CREA) were measured using a full-automatic biochemical analyzer, respectively.
As a result: according to the analysis of the behavioral results, when the administration dosage is 25-100g/kg, the bupleurum petroleum ether part has obvious improvement effect on the behavioral indexes of the CUMS model rat, shows stronger antidepressant activity and does not cause the abnormality of the liver biochemical indexes of the CUMS model rat. For normal rats, the analysis result of biochemical indexes shows that the normal rats show liver injury when the administration dose is more than or equal to 25g/kg, and show renal toxicity when the administration dose of bupleurum petroleum ether part is 100 g/kg.
2. Sample treatment: after thawing the liver tissue at 4 deg.C, precisely weighing 250mg of liver, adding 1500. mu.L of precooled 0.2% formic acid acetonitrile, homogenizing in ice, centrifuging at 13000rpm for 15min at 4 deg.C, taking the supernatant, and drying in a vacuum freeze centrifugal drier. Add 500. mu.L of 0.1% formic acid water acetonitrile (9: 1), redissolve, centrifuge at 13000rpm for 15min at 4 ℃ and the supernatant is ready for LC-MS analysis.
UHPLC-QOxactve Orbitrap-MS assay
The instrument comprises the following steps: UltMate 3000 ultra-high performance liquid chromatography tandem quadrupole electrostatic field Orbitrap mass spectrometer (UHPLC-QOxactive Orbitrap-MS, Thermo-Fisher Scientific, USA); xcalibur workstation (ThermoFisher Scientific, USA).
Chromatographic conditions are as follows: liquid phase conditions: mobile phase: a (water, 0.1% formic acid), B (acetonitrile, 0.1% formic acid); gradient of mobile phase: 0-2 min, 2% B; 2-3 min, 2-35% of B; 3-17 min, 35-70% B; 17-18 min, 70% B; 18-29 min, 70-98% B; 29-31 min, 98% B; 31-33 min, 98-2% B; 33-35 min, 2% B. Flow rate of 0.2ml/min, 5. mu.L sample size, column temperature of 40 ℃, Waters ACQUITYUPLC HSS T3 chromatography column (2.1X 100mm, 1.7 μm).
Mass spectrum conditions: adopting an ESI electrospray ionization mode; switching an acquisition mode by positive and negative ions, wherein the scanning mode comprises the following steps: FullScan/dd-MS2, wherein the m/z collection range is 100-1500. The positive electrode of the spraying voltage is 3.5 kV; the negative electrode was 2.5 kV. The capillary temperature was 320 ℃; the temperature of the heater is 300 ℃; flow rate of sheath gas: 35arb, assist gas flow rate: 10 arb; resolution was set at MS FullScan 35000 FWHM and MS/MS 17500FWHM with NCE set at 12.5eV, 25eV and 37.5 eV.
And (3) measuring results: and importing the collected LC-MS/MS data file into Compound discover 2.0 software (Thermo Fisher, USA) to acquire matched and aligned peak data, wherein the set parameters are as follows: mass range (mass): 100-1500 Da; mass deviation (MassTolerance): 5 ppm; RT tolerance [ min ]: 0.05; signal-to-noise threshold (S/NThreshold): 1.5. and introducing the peak data containing the retention time, the molecular formula, the accurate molecular weight and the peak area information into Excel for peak area normalization storage. And finally, introducing the data after peak area normalization into SIMCA-P13.0 (Umetrics, Sweden) to perform partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA). P <0.05 screen combined with VIP >1 in S-plot and t-test of independent samples simultaneously contributed the most contributing differential variable.
As shown in fig. 1, it can be seen from the dose-effect profiles (K, M, C4, C6 and C7 groups) that the CUMS model rats are clearly separated from the blank control group, and the administered group can recall metabolites to the blank control group along the T1 axis and has a clear dose dependency. The dose-toxic profile (K, Z4, Z6 and Z7 groups) clearly shows a gradual deviation from the normal group with increasing doses administered. Metabolic profiling the modulating effect of the drug on the CUMS rat metabolome and the deviating effect on normal rats can be seen visually. However, although such a visual observation method can be used for qualitative evaluation of quantitative-effect, the result evaluation is subjective. And the results are complicated, and the main quantity-effect/toxicity markers cannot be defined.
The data obtained may be semi-quantitative data or may be absolute quantitative data such as peak area, peak height, or data calculated mathematically therefrom.
4. Screening of agent with difference of amount, effect and toxicity: the differential variables screened in 3 were identified, peak areas and corresponding dose information were introduced into SPSS for pearson correlation analysis, and differential metabolites with strong correlation were screened (fig. 2). As can be seen from FIG. A, the correlation and significance of the callback differential metabolite and the administered dose, and FIG. B is a dose-response curve of each callback differential metabolite, wherein the amount-difference metabolites L-Isoleucine/leucine, L-Carnitine, L-Acetylcarnitine, Adenine and 2-Phenylacetamide, the amount-difference metabolites L-Carnitine, L-Valine, Malic acid, Adenosine 2' -phosphate and Ascorbic acid-2-sulfate are screened according to the significance P <0.001 and the correlation R > 0.6.
5. Calculation of the dose-effect/toxicity index: calculating the effect value (EI) and Toxicity value (Toxicity Index, TI) by using a mathematical model:
ki represents the average value of the content of a certain metabolite in the blank group; mi represents the average value of the corresponding metabolite content in the model group; ci represents the content of the corresponding metabolite in the model administration group. Zi represents the content of corresponding metabolite in the blank medicine group.
The pharmacodynamic index (EI) is the sum of the drug-to-metabolite callback after administration, and the larger the value, the more obvious the drug-to-metabolite callback degree is. In table 2, the calculation of dose-effect indexes of dose-effect markers of the C4 group is mainly performed, and it can be seen in table 2 that the relative contents of metabolites in rats in the model administration group are all between those in the normal group and the model group, which indicates that dose-effect markers have different degrees of callback after administration; the EI value indicates the effect value of the drug in different rats, wherein the effect value of C4-6 in the C4 group is 3.33, indicating that the degree of retrogradation in the C4 group is the best. In table 3, it can be seen that the relative contents of metabolites in rats in the normal administration group deviate from those in the normal group, indicating that the toxic marker deviates in vivo after administration; the TI value indicates the toxicity of the drug in different rats, with the Z4-5 effect value of 1.94 in the Z4 group, indicating that the deviation in the Z4 group is greatest.
Table 2 shows the calculation examples of the dose-effect index of group C4
Table 3 shows an example of calculation of the toxicity index of Z4 group
6. Dose effect/toxicity curve plotting: the dose effect/toxicity index obtained in step 5 was subjected to linear regression analysis to obtain a dose effect/toxicity curve (fig. 3). FIG. 3 (A) shows that the EI values of groups C4, C6 and C7 increased with increasing dose, indicating that the therapeutic effect of the drug increased with higher doses; the regression coefficient of 0.995 indicates that the dose-effect index can be well applied to the evaluation of the dose-effect relationship (C) of the bupleurum petroleum ether, and fig. 3 (B) can show that the TI of the groups Z4, Z6 and Z7 increases with the increase of the dose, which indicates that the in vivo metabolite level deviates from normal as the drug dose increases; the regression coefficient of 0.952 shows that the quantitative toxicity index can be well applied to the quantitative toxicity relationship (D) of the bupleurum petroleum ether.
Claims (10)
1. A combined marker for detecting the relation between antidepressant dose-effect and dose-toxicity of bupleurum petroleum ether parts is characterized in that: the quantity-effect markers include: l-isoleucine/leucine, L-carnitine, L-acetyl-carnitine, adenine, 2-phenylacetamide; the quantity-toxicity markers include: l-carnitine, valine, malic acid, 2' -adenosine phosphate, and 2-ascorbic acid sulfate.
2. The combined marker for detecting the relation between the antidepressant dose-effect and dose-toxicity of the bupleurum petroleum ether part according to claim 1, is characterized in that: the screening method of the combined marker comprises the following steps: detecting the metabolite change in the liver sample before and after normal rats and CUMS model rats are given bupleurum petroleum ether parts by using a high-resolution mass spectrometry technology, screening differential metabolites, selecting quantity-effect and quantity-toxicity metabolites to obtain a quantity-effect and quantity-toxicity marker combination, and screening out the combined marker based on the calculation of the quantity-effect and quantity-toxicity indexes of a mathematical model.
3. The combined marker for detecting the relation between the antidepressant dose-effect and dose-toxicity of the bupleurum petroleum ether part according to the claim 2, is characterized in that: the specific screening method comprises the following steps:
(1) unpredictable stress rats, namely CUMS model rats and normal rats are treated by the drug at the part of the bupleurum petroleum ether, and the relationship between the drug quantity-effect and the drug quantity-toxicity is determined according to the behavioral test and the biochemical index test;
(2) respectively detecting metabolite analysis in the biological samples before and after the drug treatment by using an analysis method of a metabonomics determination technology; carrying out metabolic profile analysis on the obtained analysis map to obtain a data set; screening potential biomarkers with antidepressant effect and toxicity by using a multivariate variable statistical analysis method for the data set; and (2) screening the quantity-effect metabolic markers and quantity-toxicity markers of the antidepressant effect and toxicity on the content change of the biomarkers in biological samples of the CUMS model rat and the normal rat by using the bupleurum petroleum ether part with the medicine effective dose gradient determined in the step (1).
4. The combination marker for detecting the relation between the antidepressant dose-effect and dose-toxicity of the bupleurum petroleum ether part according to the claim 3, is characterized in that: the biological sample is: liver, whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears, sweat, tissue, cells, or cell culture fluid.
5. The combination marker for detecting the relation between the antidepressant dose-effect and dose-toxicity of the bupleurum petroleum ether part according to the claim 3, is characterized in that: in the step (1): in the medical treatment of the bupleurum petroleum ether part, the medicine dose is as follows: the crude drugs are respectively 1, 3, 6, 12.5, 25, 50 and 100 g/kg; the administration dose of each group of rats is 10 mL/kg/d; the behavioral test was: weighing, open field experiments and sweet water preference experiments; the biochemical index test is as follows: alanine aminotransferase ALT, aspartate aminotransferase AST, alkaline phosphatase ALP, total bilirubin TBIL, urea nitrogen BUN and creatinine CREA were measured with a full-automatic biochemical analyzer, respectively.
6. The combination marker for detecting the relation between the antidepressant dose-effect and dose-toxicity of the bupleurum petroleum ether part according to the claim 3, is characterized in that: the metabonomics determination technology in the step (2) is liquid chromatogram-high resolution mass spectrum UHPLC-Q-Orbitrap-MS, a mass spectrum determination technology or a nuclear magnetic resonance determination technology.
7. The combined marker for detecting the relation between the antidepressant dose-effect and dose-toxicity of the bupleurum petroleum ether part according to claim 6, which is characterized in that: in the step (2), UHPLC-QOxacttive orbit-MS is adopted for determination, and the specific method comprises the following steps: chromatographic conditions are as follows: liquid phase conditions: mobile phase: a: water, containing 0.1% formic acid, B: acetonitrile, containing 0.1% formic acid; gradient of mobile phase: 0-2 min, 2% B; 2-3 min, 2-35% of B; 3-17 min, 35-70% B; 17-18 min, 70% B; 18-29 min, 70-98% B; 29-31 min, 98% B; 31-33 min, 98-2% B; 33-35 min, 2% B; flow rate of 0.2ml/min, 5 μ L sample size, column temperature 40 ℃, watersaccquityuplc HSS T3 chromatography column: 2.1 × 100mm, 1.7 μm;
mass spectrum conditions: adopting an ESI electrospray ionization mode; switching an acquisition mode by positive and negative ions, wherein the scanning mode comprises the following steps: FullScan/dd-MS2, wherein the m/z acquisition range is 100-1500; the positive electrode of the spraying voltage is 3.5 kV; the negative electrode is 2.5 kV; the capillary temperature was 320 ℃; the temperature of the heater is 300 ℃; flow rate of sheath gas: 35arb, assist gas flow rate: 10 arb; resolution was set at MS FullScan 35000 FWHM and MS/MS 17500FWHM, NCE was set at 12.5eV, 25eV and 37.5 eV;
the specific test method for the obtained LC-MS spectrum data comprises the following steps: and importing the acquired LC-MS/MS data file into Compound discover 2.0 software to acquire matched and aligned peak data, wherein the set parameters are as follows: mass range (mass range): 100-1500 Da; mass deviation (MassTolerance): 5 ppm; RT tolerance [ min ]: 0.05; signal-to-noise threshold (S/NThreshold): 1.5;
leading the peak data containing the retention time, molecular formula, accurate molecular weight and peak area information into Excel for peak area normalization storage; finally, the data after the peak area normalization is led into SIMCA-P13.0 to carry out partial least square discriminant analysis PLS-DA and orthogonal partial least square discriminant analysis OPLS-DA; simultaneously screening the difference variable with the largest contribution by combining VIP >1 in an S-plot and P <0.05 of t test of an independent sample;
and identifying the screened differential variables, introducing the peak areas and corresponding dosage information into SPSS (SpSS) for carrying out Pearson correlation analysis, and screening out differential metabolites with strong correlation.
8. The combination marker for detecting the relation between the antidepressant dose-effect and dose-toxicity of the bupleurum petroleum ether part according to the claim 3, is characterized in that: the specific dose-effect/toxicity index relation calculation adopts a pharmacodynamic index EI mathematical model, partial least square-discriminant analysis PLS-DA, orthogonal partial least square method OPLS, orthogonal partial least square method-discriminant analysis OPLS-DA, principal component analysis PCA, nonlinear mapping NLM or cluster analysis HCA mathematical model.
9. The combined marker for detecting the relation between the antidepressant dose-effect and the dose-toxicity of the bupleurum petroleum ether part according to claim 8, is characterized in that the calculation of the specific dose-effect/toxicity index relation adopts a mathematical model, and the calculation method comprises the following steps: the calculation formula of the effect value EI and the toxicity value TI is as follows:
wherein: ki represents the average value of the content of a certain metabolite in the blank group; mi represents the average value of the corresponding metabolite content in the model group; ci represents the content of corresponding metabolites in the model administration group; zi represents the content of corresponding metabolite in the blank administration group; and carrying out linear regression analysis on the dose effect/toxicity index to obtain a dose effect/toxicity curve.
10. Use of a combination marker according to claim 1, characterized in that: the combined marker is used for evaluating the relation between the depression resisting amount and the effect/toxicity of the bupleurum.
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