阅读说明：本技术 一种评价中成药药代基质效应的分析方法 (Analysis method for evaluating matrix effect of Chinese patent medicine ) 是由 李川 程晨 余玄 杜飞飞 徐方 王凤清 董凯 张桂萍 姚小青 于 2019-11-01 设计创作，主要内容包括：本发明提供一种评价中成药药代基质效应的分析方法,包括以下步骤：1)将中成药和中成药中关键成分分别对实验鼠给药后,采集体液；2)对体液进行分析,确定体液所含中成药中关键成分的浓度；3)采用梯形法计算获得AUC<Sub>0-t</Sub>；4)计算AUC<Sub>0-∞</Sub>；5)计算血浆总清除率；6)计算肾清除率、胆汁清除率；7)计算稳态分布容积；8)计算尿累积排泄分数、胆汁累积排泄分数；9)将药代动学参数,进行样本T检验评价显著性差异,确定中成药中关键成分的药代基质效应。本发明提供的一种评价中成药药代基质效应的分析方法,可以快速准确地评价中成药关键成分的药代基质效应,对于揭示基于药代的中成药中关键成分方剂配伍规律具有重要意义。(The invention provides an analysis method for evaluating the effect of a Chinese patent drug-substituted matrix, which comprises the following steps: 1) respectively administering key components of the Chinese patent medicine and the Chinese patent medicine to a laboratory mouse, and collecting body fluid; 2) analyzing the body fluid to determine the concentration of key components in the Chinese patent medicine contained in the body fluid; 3) AUC is obtained by trapezoidal calculation 0‑t (ii) a4) Calculating AUC 0‑∞ (ii) a 5) Calculating the total clearance rate of the blood plasma; 6) calculating renal clearance and bile clearance; 7) calculating a steady-state distribution volume; 8) calculating cumulative urine excretion fraction and cumulative bile excretion fraction; 9) and (3) carrying out sample T test on pharmacokinetic parameters to evaluate the significance difference, and determining the drug-induced matrix effect of key components in the Chinese patent medicine. The invention provides a commentThe analysis method of the drug-substituted matrix effect of the Chinese patent medicine can quickly and accurately evaluate the drug-substituted matrix effect of the key components of the Chinese patent medicine, and has important significance for disclosing the compatibility rule of key component prescriptions in the Chinese patent medicine based on the drug substitution.)

1. An analysis method for evaluating the effect of a Chinese patent drug-substituted matrix comprises the following steps:

1) respectively taking the key components in the Chinese patent medicine and the Chinese patent medicine to the laboratory mice, and collecting body fluids of the laboratory mice taking the key components in the Chinese patent medicine and the Chinese patent medicine at different time points t to respectively serve as a sample group A and a sample group B;

2) analyzing the sample group A and the sample group B by adopting a liquid chromatography-mass spectrometry combined method, and determining the concentration C of key components in the Chinese patent medicines contained in the sample group A and the sample group B at different time points t_t；

3) According to the concentration C_tLinear relation with corresponding time point t, and trapezoidal method is adopted to calculate and obtain area AUC_0-t；

4) Calculating AUC_0-∞So that it complies with formula (1), said formula (1) being: AUC_0-∞＝AUC_0-t+C_t/k_eWherein AUC_0-∞The area under the curve of the time of the drug from 0 moment to infinity, mu M.h; AUC_0-tThe area under the curve of the time of administration, μ M · h, over the period of time from 0 moment before administration to the last time point t selected experimentally; c_tIs the concentration of key components in the Chinese patent medicine contained in the body fluid of the experimental mouse at different time points t, namely mu M; k is a radical of_eFor the end elimination rate, h^-1；

5) According to AUC_0-∞Calculating the total clearance CL of plasma according to the formula (2)_tot,p；

The formula (2) is: CL_tot,p＝Dose/AUC_0-∞Wherein, CL is_tot,pThe total clearance of the blood plasma is L/h/kg; dose is Dose, μmol; AUC_0-∞The area under the curve of the time of the drug from 0 moment to infinity, mu M.h;

6) according to AUC_0-tCalculating the renal clearance CL according to the formula (3)_RCalculating bile clearance CL according to equation (4)_B；

The formula (3) is: CL_R＝Cum.A_e-U/AUC_0-tWherein, CL is_RL/h/kg for renal clearance; Cum.A_e-Uμ mol to accumulate urine excretion; AUC_0-tFor the period of time from 0 before the administration to the last time point t selected for the experimentArea under the curve of the time curve in the middle, μ M · h;

the formula (4) is: CL_B＝Cum.A_e-B/AUC_0-tWherein, CL is_BThe bile clearance is L/h/kg; Cum.A_e-Bμ mol to accumulate bile excretion; AUC_0-tThe area under the curve of the time of administration, μ M · h, over the period of time from 0 moment before administration to the last time point t selected experimentally;

7) according to CL_tot,pCalculating the steady-state distribution volume V according to the formula (5)_ss，

The formula (5) is: v_ss＝CL_tot,p*t_1/2Wherein V is_ssIs a steady distribution volume, L/kg; CL_tot,pThe total clearance of the blood plasma is L/h/kg; t is t_1/2To eliminate half-life, h;

8) calculating cumulative urine voiding fraction f according to equation (6)_e-UCalculating the cumulative bile excretion fraction f according to the formula (7)_e-B；

The formula (6) is: f. of_e-U＝Cum.A_e-U100% of/Dose, wherein f_e-UCumulative urine excretion fraction,%; Cum.A_e-Uμ mol to accumulate urine excretion; dose is Dose, μmol;

the formula (7) is: f. of_e-B＝Cum.A_e-B100% of/Dose, wherein f_e-BCumulative bile excretion fraction,%; Cum.A_e-Bμ mol to accumulate bile excretion; dose is Dose, μmol;

9) in sample group A and sample group B, C_t、AUC_0-t、AUC_0-∞CL as a dose-dependent plasma pharmacokinetic parameter_tot,p、V_ssAs a dose-independent plasma pharmacokinetic parameter, f was chosen_e-U、f_e-B、CL_R、CL_BOne or more of the above-mentioned ingredients are used as pharmacokinetic parameters for reflecting excretion, and the sample T test is carried out to evaluate the significant difference, so as to determine the drug-induced matrix effect of key ingredients in the Chinese patent medicine.

2. The method as claimed in claim 1, wherein in step 1), the body fluid of the laboratory mouse is selected from one or more of plasma, urine and bile.

3. The assay method for evaluating the effect of a Chinese patent drug-substrate according to claim 2, wherein when the body fluid is plasma, the time t is any time point before administration and 0, 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24h after administration; when the body fluid is urine or bile, the time points t are 0-12h before administration and 0-4, 4-8 and 8-24 h after administration.

4. The method as claimed in claim 1, wherein in step 2), the body fluid of the laboratory mouse is added with solvent before analysis, and then shaken for protein precipitation and centrifugation, and the supernatant is taken as the analysis solution.

5. The assay method for evaluating the effect of a Chinese patent drug-substrate according to claim 4, which comprises any one or more of the following conditions:

A1) the solvent is methanol;

A2) the volume ratio of the body fluid of the experimental mouse to the solvent is 1: 2-4;

A3) the oscillation time is 1-6 min;

A4) the centrifugation conditions were: the centrifugation speed is 10000-; the centrifugation time is 5-15 min.

6. The analytical method for evaluating the effect of a Chinese patent drug-substituted matrix according to claim 1, wherein in the step 2), the liquid chromatography-mass spectrometry is performed by using a Water MG II column, 50mm x 2.0mm, 3 μm; the temperature of the chromatographic column is room temperature; the flow rate is 0.2-0.4 mL/min; the sample amount is 3-10 μ L.

7. The analytical method for evaluating the effect of a Chinese patent drug-substituted matrix according to claim 1, wherein in the step 2), the liquid chromatography-mass spectrometry is performed by using the following mobile phases: water-methanol solution containing formic acid, wherein phase A is water containing 0.003-0.005% formic acid; phase B is methanol containing 0.003-0.005% formic acid; analysis time: 10 min; gradient elution.

8. The analysis method for evaluating the effect of a Chinese patent drug-substituted matrix according to claim 7, wherein the gradient elution procedure comprises: 0-0.5min, phase A: the volume ratio of the phase B is 98: 2-98: 2; 0.5-7min, phase A: the volume ratio of the phase B is 98: 2-30: 70; 7-8min, phase A: the volume ratio of the phase B is 30: 70-2: 98, respectively; 8-8.1min, phase A: the volume ratio of the phase B is 2: 98-98: 2; 8.1-10min, phase A: the volume ratio of the phase B is 98: 2-98: 2.

9. the analytical method for evaluating the effect of a Chinese patent drug-substituted matrix according to claim 1, wherein in the step 2), the measurement conditions of the mass spectrum in the liquid chromatography-mass spectrometry are as follows: the ion source is an ESI source; the detection mode is an anion electrospray ionization mode ESI^-(ii) a The working mode is an SRM mode; the SRM ion pairs used for quantitative analysis were: m/z 611 → 491.

10. The analysis method for evaluating the effect of a Chinese patent drug-substrate according to claim 1, wherein in step 9), the sample T test is calculated according to the formula (8), and the formula (8) is:

wherein the content of the first and second substances,is the mean of two samples; sigma_x1 ²、σ_x2 ²The two recipes are different; gamma is the correlation coefficient of the correlation sample;

n is the sample size.

Technical Field

The invention belongs to the field of pharmacokinetics of traditional Chinese medicines, and relates to an analysis method for evaluating the effect of a Chinese patent medicine matrix.

Background

The Chinese patent medicine is a commercialized Chinese medicinal preparation approved by the national drug supervision and management department, and is prepared into a Chinese medicinal product with a certain dosage form according to a specified prescription and a preparation process under the guidance of the theory of traditional Chinese medicine and in order to prevent and treat diseases. The Chinese patent medicine can be prepared into tablets, pills, powder, paste, injection and the like. Compared with chemical medicines, the Chinese patent medicine has complex chemical components and complex change process in a body after administration. The matrix effect of Chinese medicine is that the single component in Chinese patent medicine is affected by other components, and compared with the single component administration, the pharmacokinetic characteristic in vivo changes. The investigation of the effect of the traditional Chinese medicine substituting matrix has important significance for clarifying the in-vivo pharmacokinetic characteristics of the traditional Chinese medicine monomer components after compound administration and revealing the prescription compatibility rule of key components. At present, no method for systematically investigating the effect of the traditional Chinese medicine substituting matrix is proposed.

Disclosure of Invention

The invention aims to provide an analysis method for evaluating the matrix effect of Chinese patent medicine, which can quickly and accurately evaluate the matrix effect of key substances of Chinese patent medicine.

In order to achieve the above objects and other related objects, the present invention provides an analysis method for evaluating the effect of a Chinese patent drug-substituted matrix, comprising the following steps:

1) respectively taking the key components in the Chinese patent medicine and the Chinese patent medicine to the laboratory mice, and collecting body fluids of the laboratory mice taking the key components in the Chinese patent medicine and the Chinese patent medicine at different time points t to respectively serve as a sample group A and a sample group B;

2) analyzing the sample group A and the sample group B by adopting a liquid chromatography-mass spectrometry combined method, and determining the concentration C of key components in the Chinese patent medicines contained in the sample group A and the sample group B at different time points t_t；

3) According to the concentration C_tLinear relation with corresponding time point t, and trapezoidal method is adopted to calculate and obtain area AUC_0-t；

4) Calculating AUC_0-∞So that it complies with formula (1), said formula (1) being: AUC_0-∞＝AUC_0-t+C_t/k_eWherein AUC_0-∞The area under the curve of the time of the drug from 0 moment to infinity, mu M.h; AUC_0-tThe area under the curve of the time of administration, μ M · h, over the period of time from 0 moment before administration to the last time point t selected experimentally; c_tIs the concentration of key components in the Chinese patent medicine contained in the body fluid of the experimental mouse at different time points t, namely mu M; k is a radical of_eFor the end elimination rate, h^-1；

5) According to AUC_0-∞Calculating the total clearance CL of plasma according to the formula (2)_tot,p；

The formula (2) is: CL_tot,p＝Dose/AUC_0-∞Wherein, CL is_tot,pThe total clearance of the blood plasma is L/h/kg; dose is Dose, μmol; AUC_0-∞The area under the curve of the time of the drug from 0 moment to infinity, mu M.h;

6) according to AUC_0-tThe renal clearance was calculated according to the formula (3)CL_RCalculating bile clearance CL according to equation (4)_B；

The formula (3) is: CL_R＝Cum.A_e-U/AUC_0-tWherein, CL is_RL/h/kg for renal clearance; Cum.A_e-Uμ mol to accumulate urine excretion; AUC_0-tThe area under the curve of the time of administration, μ M · h, over the period of time from 0 moment before administration to the last time point t selected experimentally;

the formula (4) is: CL_B＝Cum.A_e-B/AUC_0-tWherein, CL is_BThe bile clearance is L/h/kg; Cum.A_e-Bμ mol to accumulate bile excretion; AUC_0-tThe area under the curve of the time of administration, μ M · h, over the period of time from 0 moment before administration to the last time point t selected experimentally;

7) according to CL_tot,pCalculating the steady-state distribution volume V according to the formula (5)_ss，

The formula (5) is: v_ss＝CL_tot,p*t_1/2Wherein V is_ssIs a steady distribution volume, L/kg; CL_tot,pThe total clearance of the blood plasma is L/h/kg; t is t_1/2To eliminate half-life, h;

8) calculating cumulative urine voiding fraction f according to equation (6)_e-UCalculating the cumulative bile excretion fraction f according to the formula (7)_e-B；

The formula (6) is: f. of_e-U＝Cum.A_e-U100% of/Dose, wherein f_e-UCumulative urine excretion fraction,%; Cum.A_e-Uμ mol to accumulate urine excretion; dose is Dose, μmol;

the formula (7) is: f. of_e-B＝Cum.A_e-B100% of/Dose, wherein f_e-BCumulative bile excretion fraction,%; Cum.A_e-Bμ mol to accumulate bile excretion; dose is Dose, μmol;

9) in sample group A and sample group B, C_t、AUC_0-t、AUC_0-∞CL as a dose-dependent plasma pharmacokinetic parameter_tot,p、V_ssAs a dose independent plasmaPharmacokinetic parameters, selection of f_e-U、f_e-B、CL_R、CL_BOne or more of the above-mentioned ingredients are used as pharmacokinetic parameters for reflecting excretion, and the sample T test is carried out to evaluate the significant difference, so as to determine the drug-induced matrix effect of key ingredients in the Chinese patent medicine.

Preferably, in the step 1), the Chinese patent medicine is a Xuebijing injection.

In the step 1), the key components in the Chinese patent medicine refer to components which can be used for evaluating the matrix effect of the Chinese patent medicine in the Chinese patent medicine.

Preferably, in step 1), the laboratory mouse is an SD rat.

Preferably, in step 1), the administration mode is selected from one of oral administration or injection. The injection is selected from one of intravenous injection, subcutaneous injection and intramuscular injection.

Preferably, in step 1), the body fluid of the laboratory mouse is one or more of plasma, urine or bile.

More preferably, when the body fluid is plasma, centrifuging and freezing to be tested after blood collection; when the body fluid is urine, collecting a urine sample, and then freezing and storing the urine sample to be tested; and when the body fluid is bile, collecting the bile, and freezing and storing the bile to be tested.

Further preferably, the conditions of the centrifugation are: the centrifugation speed is 5000-; the centrifugation time is 1-3 min. Most preferably, the conditions of the centrifugation are: the centrifugation speed is 10000 rpm; the centrifugation time was 2 min.

Further preferably, the temperature for cryopreservation is ≦ -70 ℃.

More preferably, when the body fluid is plasma, the time point t is any time point before administration and 0, 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24h after administration.

More preferably, when the body fluid is urine or bile, the time points t are 0 to 12 hours before administration and 0 to 4, 4 to 8 and 8 to 24 hours after administration.

Preferably, in step 1), the experimental mice can be taken with water but fasted for 10-14h before administration. More preferably, the experimental mice are fasted for 12h before administration.

Preferably, in step 2), the body fluid of the laboratory mouse is added with a solvent before analysis, and then is shaken to perform protein precipitation and centrifugation, and the supernatant is taken as an analysis solution.

More preferably, the solvent is methanol.

More preferably, the ratio of the body fluid of the laboratory mouse to the volume of the solvent added is 1: 2-4. Most preferably, the ratio of the volume of body fluid to solvent added to the laboratory rat is 1: 3.

More preferably, the oscillation time is 1-6 min. Most preferably, the oscillation time is 5 min.

More preferably, the conditions of the centrifugation are: the centrifugation speed is 10000-; the centrifugation time is 5-15 min. Most preferably, the conditions of the centrifugation are: the centrifugation rate is 14800 rpm; the centrifugation time was 10 min.

Preferably, in the step 2), the liquid chromatography-mass spectrometry combination method adopts a Water MG II column (50mm × 2.0mm, 3 μm).

Preferably, in the step 2), in the liquid chromatography-mass spectrometry combined method, the temperature of a chromatographic column used for liquid chromatography is room temperature, and preferably is 20-25 ℃; the flow rate is 0.2-0.4ml/min, preferably 0.3 ml/min; the amount of sample is 3-10. mu.L, preferably 5. mu.L.

Preferably, in the step 2), in the liquid chromatography-mass spectrometry combination method, the mobile phase adopted by the liquid chromatography is as follows: water-methanol solution (containing formic acid), wherein phase A is water (containing 0.003-0.005% formic acid), and phase B is methanol (containing 0.003-0.005% formic acid); analysis time: 10 min; gradient elution.

More preferably, in the liquid chromatography-mass spectrometry combined method, the liquid chromatography adopts the following mobile phases: water-methanol solution (containing formic acid), wherein phase A is water (containing 0.004% formic acid) and phase B is methanol (containing 0.004% formic acid); analysis time: 10 min; gradient elution.

More preferably, the gradient elution procedure is specifically:

0-0.5min, phase A: the volume ratio of the phase B is 98: 2-98: 2; 0.5-7min, phase A: the volume ratio of the phase B is 98: 2-30: 70; 7-8min, phase A: the volume ratio of the phase B is 30: 70-2: 98, respectively; 8-8.1min, phase A: the volume ratio of the phase B is 2: 98-98: 2; 8.1-10min, phase A: the volume ratio of the phase B is 98: 2-98: 2.

preferably, in the step 2), in the liquid chromatography-mass spectrometry combination method, the determination conditions of the mass spectrum are as follows: the ion source is an ESI source; the detection mode is anion electrospray ionization (ESI)^-) (ii) a The working mode is an SRM mode; the SRM ion pairs used for quantitative analysis were: m/z 611 → 491.

Preferably, in the step 2), the liquid chromatography-mass spectrometry is used for analyzing the body fluid of the laboratory mouse, and the concentration of the key component in the corresponding Chinese patent medicine contained in the body fluid of the laboratory mouse is determined qualitatively by mass spectrometry according to the key component standard in the Chinese patent medicine with known concentration, and then quantified by a standard curve method.

The standard curve method is characterized in that a corresponding standard working curve is drawn through a linear relation between chromatographic peak areas of key components in the Chinese patent medicines obtained through LC/MS/MS measurement and concentrations of the key components in the corresponding Chinese patent medicines, a regression equation of the standard working curve of the key components in the Chinese patent medicines is obtained through calculation, chromatographic peak areas of the key components in the corresponding Chinese patent medicines contained in body fluid of the experimental mouse are measured through LC/MS/MS measurement and substituted into the regression equation of the standard working curve of the key components in the corresponding Chinese patent medicines, and the concentrations of the key components in the corresponding Chinese patent medicines contained in the body fluid of the experimental mouse are obtained through calculation.

Preferably, in the step 2), the number n of key components in the Chinese patent medicine is a natural number not less than 1. More preferably, n is a natural number of 1 to 6.

In the step 3), the trapezoidal method is to adjust the blood concentration C_tThe time t curve is divided into countless small trapezoids according to the area formula of the trapezoids: multiplying (upper bottom + lower bottom) by the height divided by 2, calculating each small trapezoid area, and adding all trapezoid areas to obtain the area under the time curve AUC_0-t. According to the concentration C_tThe concentration C is plotted as a linear relationship with the corresponding time point t_tCurve with time point t.

In step 4), the AUC (area under the curve at time of drug administration) represents the total amount of drug entering the systemic circulation after administration, reflecting the plasma exposure level of the drug.

In step 4), the terminal elimination rate k_eComprises the following steps: blood concentration C_tTaking logarithm, and performing linear regression on the time t to obtain the negative number of the slope value. The end cancellation rate k_eFitting calculations were performed by Kinetica5.0 software.

In step 5), the CL_tot,pIs the sum of the clearance rates of the drugs of liver and kidney, i.e. how much volume of plasma is cleared of the drugs per unit time, and this parameter reflects the ability of the body to clear the drugs.

In step 6), the CL_RThe ability of kidney to expel drug from plasma within a certain period of time reflects the ability of kidney to clear drug on one hand and the mechanism of drug clearance from the body on the other hand, and is one of the components of the total clearance of drug plasma.

In step 6), the CL_BThe ability of the liver to discharge the drug in the plasma out of the body in a certain time by means of bile excretion reflects the clearance of the liver to the drug on the one hand and the clearance mechanism of the drug from the body on the other hand, and is one of the components of the total clearance rate of the drug plasma.

In step 7), the V_ssThe pharmacological meaning of the ratio of the drug amount in vivo to the blood concentration after the drug achieves dynamic balance in vivo is to indicate whether the drug is widely distributed in vivo or the drug is greatly combined with a biopolymer or both.

In step 7), the t_1/2(elimination half-life, also called terminal elimination half-life) is calculated from the terminal elimination phase of the time curve, and is numerically related to the terminal elimination rate k_eInversely proportional, i.e. terminal elimination half-life t_1/2＝0.693/k_e(ii) a The parameter is the time required for the end phase blood concentration to be reduced by half, and the elimination speed of the medicine from the body is intuitively reflected.

In step 8), f is_e-UReflecting the extent of excretion of the drug from the urine.

In step 8), f is_e-BReflecting the extent of excretion of the drug from the bile.

Step 6) or8) In the formula (3) or (6), the cumulative urine excretion cum.a_e-UIs the sum of the product of the concentration of key components in the Chinese patent medicine in urine and the excretion volume in each time period.

Step 6) or 8), the cumulative bile excretion cum.a in the formula (4) or (7)_e-BIs the sum of the products of the concentration of key components in the Chinese patent medicine in the bile and the excretion volume in each time period.

In the step 9), the sample T is tested, and aims to investigate whether the in vivo pharmacokinetic characteristics of the key components in the selected Chinese patent medicine to be tested have significant differences in two administration modes of the sample group a and the sample group B, if the differences significantly indicate that other components in the Chinese patent medicine may affect the in vivo pharmacokinetic characteristics of the key components in the selected Chinese patent medicine to be tested, the in vivo pharmacokinetic characteristics of the key components in the Chinese patent medicine to be tested may have a traditional Chinese medicine pharmacokinetic matrix effect, and if no significant differences indicate that other components in the Chinese patent medicine do not affect the in vivo pharmacokinetic characteristics of the key components in the Chinese patent medicine to be tested, the in vivo pharmacokinetic characteristics of the key components in. Preferably, in step 9), the sample T test is calculated according to formula (8), where formula (8) is:

wherein the content of the first and second substances,

is the mean of two samples; sigma_x1 ²、σ_x2 ²The two recipes are different; gamma is the correlation coefficient of the correlation sample; n is the sample size. The equation (8) is calculated by using SPSS19.0 software, wherein the correlation coefficient γ of the correlation sample is calculated by fitting the SPSS19.0 software.

Preferably, the sample T test indicates a statistically significant difference when P < 0.05.

In the steps 4), 5), 6), 7) or 8), the pharmacokinetic parameters in the formulas (1) to (7) can be analyzed by using pharmacokinetic software Kinetica5.0 according to a non-compartmental model method for data processing.

As described above, the analysis method for evaluating the pharmacokinetic matrix effect of the Chinese patent drug provided by the invention comprises the steps of respectively administering key monomer components in the Chinese patent drug and the Chinese patent drug to a laboratory mouse, collecting body fluid of the laboratory mouse, measuring the concentration of the key monomer components existing in the body fluid of the laboratory mouse, calculating pharmacokinetic parameters of obtained data, carrying out statistical analysis, and comparing the in-vivo processes (blood concentration, urine and/or bile excretion and excretion rate) of the key substances in the Chinese patent drug compound preparation administration and the Chinese patent drug after the key substances in the Chinese patent drug are independently administered, thereby revealing whether the in-vivo processes of the key components in the Chinese patent drug after the Chinese patent drug administration are influenced by other components, whether the in-vivo pharmacokinetic characteristics are obviously changed, namely whether the pharmacokinetic matrix effect of the Chinese patent drug is generated. The method can quickly and accurately evaluate the matrix effect of the key components in the Chinese patent medicine, and has important significance for disclosing the compatibility rule of the Chinese medicinal prescription based on the generation.

Drawings

FIG. 1 is a graph showing the mean blood concentration of hydroxysafflor yellow A in rats after administration of a Xuebijing injection according to the present invention, plotted against time.

FIG. 2 is a graph showing the mean blood concentration of hydroxysafflor yellow A in rats after administration of the hydroxysafflor yellow A solution of the present invention versus time.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The reagents and equipment used in the following examples are as follows:

1. reagent

SPF-grade, male, weight 230-; use of experimental animals in the animal center of Shanghai pharmaceutical research institute of Chinese academy of sciences stage II [ the use of experimental animals license number: SYXK (Shanghai) 2015-0027); the laboratory protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of shanghai pharmaceutical research institute of china academy of sciences before implementation (2012-11-LC-08).

A hydroxysafflor yellow A (hereinafter referred to as hydroxyA, CAS No. 78281-02-4) reference was purchased from Shidan Biotechnology Ltd; xuebijing injection (Xuebijing, the Chinese medicine standard: Z20040033) is provided by Tianjin Hongyi pharmaceutical industry, Inc.

Methanol, formic acid (LC-MS grade reagent, Merck, germany); the experimental water was ultrapure water (18.2 M.OMEGA.) prepared by a Millipore Mill-QIntegral3 system.

2. Instrument for measuring the position of a moving object

The liquid phase-mass spectrometry system (LC/MS/MS) consists of a Waters UPLC and a TSQQuantum triple quadrupole mass spectrometer detector manufactured by Thermo corporation, and the system working software is Chemstation (HPLC) and Xcalibur 1.3 (mass spectrum).

Ultra low temperature refrigerator at-70 deg.C (Sanyo Co., Japan); sorvall Legend RT Low temperature bench centrifuge (Germany); vibrax VXR mini shaker (germany); USC302 ultrasonic cleaner (shanghai polarone electronics limited).

In one embodiment, the analysis method for evaluating the effect of the drug-matrix of the Chinese patent medicine comprises the following analysis processes.

SD rats are selected as experimental animals, and the SD rats are selected from SPF grade, male and 230-270 g in body weight. The Chinese patent medicine compound components which are used as key components in the Chinese patent medicines and the Chinese patent medicines are respectively administrated to the experimental mice, the administration mode is selected from one of oral administration or injection, and the injection is selected from one of intravenous injection, subcutaneous injection and intramuscular injection. The experimental mice can be fed with water but fasted for 10-14h before administration.

Then, body fluids of the rats to which the Chinese patent drugs were administered and key ingredients in the Chinese patent drugs were administered were collected at different time points t and used as a sample group a and a sample group B, respectively. The body fluid of the experimental mouse is one or more of plasma, urine or bile. When the body fluid is plasma, centrifuging at 5000-. When the body fluid is urine, after collecting urine sample, freezing and storing at ≦ -70 deg.C for testing, and selecting time points t of 0-12h before administration and 0-4, 4-8 and 8-24 h after administration. When the body fluid is bile, collecting bile, and freezing and storing at-70 deg.C or below for testing, wherein the selected time t is 0-12h before administration and 0-4, 4-8 and 8-24 h after administration.

Analyzing the body fluid of the experimental mouse by adopting a liquid chromatography-mass spectrometry combined method, and determining the concentration C of key components in n Chinese patent medicines contained in the body fluid of the experimental mouse at different time points t_t. Wherein n is a natural number not less than 1. Adding methanol before analysis, oscillating for 1-6min for protein precipitation, centrifuging at 10000-. The ratio of the body fluid of the experimental mouse to the volume of the methanol is 1: 2-4.

The liquid chromatography-mass spectrometry combined method is used for analyzing the body fluid of the laboratory mouse, firstly, the mass spectrometry is used for determining the quality of the key components in the Chinese patent medicines with known concentrations, and then, the standard curve method is used for quantifying the concentration of the key components in the corresponding Chinese patent medicines in the body fluid of the laboratory mouse. The standard curve method is characterized in that a linear relation between the chromatographic peak area of key components in the Chinese patent medicine obtained through LC/MS/MS measurement and the concentration of the key components in the corresponding Chinese patent medicine is used for drawing a corresponding standard working curve, a regression equation of the standard working curve of the key components in the Chinese patent medicine is obtained through calculation, the chromatographic peak area of the key components in the corresponding Chinese patent medicine contained in the body fluid of the laboratory mouse is measured through LC/MS/MS measurement and substituted into the regression equation of the standard working curve of the key components in the corresponding Chinese patent medicine, and the concentration of the key components in the corresponding Chinese patent medicine contained in the body fluid of the laboratory mouse is obtained through calculation.

The liquid chromatography adopts Water MG II column (50mm × 2.0mm, 3 μm), and the column temperature is room temperature; the flow rate is 0.2-0.4 mL/min; the sample amount is 3-10 μ L. Mobile phase used for liquid chromatography: water-methanol solution (containing formic acid), wherein phase A is water (containing 0.003-0.005% formic acid), and phase B is methanol (containing 0.003-0.005% formic acid); analysis time: 10 min; gradient elution.

The gradient elution procedure is specifically as follows: 0-0.5min, phase A: the volume ratio of the phase B is 98: 2-98: 2; 0.5-7min, phase A: the volume ratio of the phase B is 98: 2-30: 70; 7-8min, phase A: the volume ratio of the phase B is 30: 70-2: 98, respectively; 8-8.1min, phase A: the volume ratio of the phase B is 2: 98-98: 2; 8.1-10min, phase A: the volume ratio of the phase B is 98: 2-98: 2.

the mass spectrum measurement conditions were: the ion source is an ESI source; the detection mode is anion electrospray ionization (ESI)^-) (ii) a The working mode is an SRM mode; the SRM ion pairs used for quantitative analysis were: m/z 611 → 491.

And analyzing pharmacokinetic parameters by using pharmacokinetic software Kinetica5.0 according to a non-compartmental model method, and processing data. Wherein, according to the concentration C_tThe concentration C is plotted as a linear relationship with the corresponding time point t_tCalculating the area AUC with trapezoidal method according to the curve of time point t_0-t. The trapezoidal method is to make the blood concentration C_tThe time t curve is divided into countless small trapezoids according to the area formula of the trapezoids: multiplying (upper bottom + lower bottom) by the height divided by 2, calculating each small trapezoid area, and adding all trapezoid areas to obtain the area under the time curve AUC_0-t。

Calculating AUC_0-∞So that it complies with formula (1), said formula (1) being: AUC_0-∞＝AUC_0-t+C_t/k_eWherein AUC_0-∞The area under the curve of the time of the drug from 0 moment to infinity, mu M.h; AUC_0-tThe area under the curve of the time of administration, μ M · h, over the period of time from 0 moment before administration to the last time point t selected experimentally; c_tIs the concentration of key components in the Chinese patent medicine contained in the body fluid of the experimental mouse at different time points t, namely mu M; k is a radical of_eFor the end elimination rate, h^-1. The AUC (area under the curve of the time of administration) represents the total amount of the drug entering the systemic circulation after administration, and reflects the blood of the drugLevel of pulp exposure.

According to AUC_0-∞Calculating the total clearance CL of plasma according to the formula (2)_tot,pThe formula (2) is: CL_tot,p＝Dose/AUC_0-∞Wherein, CL is_tot,pThe total clearance of the blood plasma is L/h/kg; dose is Dose, μmol; AUC_0-∞The area under the curve of the drug time from 0 to infinity, μ M · h. The CL_tot,pIs the sum of the clearance rates of the drugs of liver and kidney, i.e. how much volume of plasma is cleared of the drugs per unit time, and this parameter reflects the ability of the body to clear the drugs.

According to AUC_0-tCalculating the renal clearance CL according to the formula (3)_RCalculating bile clearance CL according to equation (4)_B。

The formula (3) is: CL_R＝Cum.A_e-U/AUC_0-tWherein, CL is_RL/h/kg for renal clearance; Cum.A_e-Uμ mol to accumulate urine excretion; AUC_0-tThe area under the time curve, μ M · h, is the time period from 0 a before the administration to the last time point t selected experimentally. The CL_RThe ability of kidney to expel drug from plasma within a certain period of time reflects the ability of kidney to clear drug on one hand and the mechanism of drug clearance from the body on the other hand, and is one of the components of the total clearance of drug plasma.

The formula (4) is: CL_B＝Cum.A_e-B/AUC_0-tWherein, CL is_BThe bile clearance is L/h/kg; Cum.A_e-Bμ mol to accumulate bile excretion; AUC_0-tThe area under the time curve, μ M · h, is the time period from 0 a before the administration to the last time point t selected experimentally. The CL_BThe ability of the liver to discharge the drug in the plasma out of the body in a certain time by means of bile excretion reflects the clearance of the liver to the drug on the one hand and the clearance mechanism of the drug from the body on the other hand, and is one of the components of the total clearance rate of the drug plasma.

According to CL_tot,pCalculating the steady-state distribution volume V according to the formula (5)_ssThe formula (5) is: v_ss＝CL_tot,p*t_1/2Wherein V is_ssIs a steady distribution volume, L/kg; CL_tot,pThe total clearance of the blood plasma is L/h/kg; t is t_1/2To eliminate half-life, h. The V is_ssThe pharmacological meaning of the ratio of the drug amount in vivo to the blood concentration after the drug achieves dynamic balance in vivo is to indicate whether the drug is widely distributed in vivo or the drug is greatly combined with the biological polymer or both. Said t is_1/2The elimination speed of the medicine from the body is visually reflected.

Calculating cumulative urine voiding fraction f according to equation (6)_e-UCalculating the cumulative bile excretion fraction f according to the formula (7)_e-B(％)。

The formula (6) is: f. of_e-U＝Cum.A_e-U100% of/Dose, wherein f_e-UCumulative urine excretion fraction,%; Cum.A_e-Uμ mol to accumulate urine excretion; dose is the Dose administered, μmol. F is_e-UReflecting the extent of excretion of the drug from the urine.

The formula (7) is: f. of_e-B＝Cum.A_e-B100% of/Dose, wherein f_e-BCumulative bile excretion fraction,%; Cum.A_e-Bμ mol to accumulate bile excretion; dose is the Dose administered, μmol. F is_e-BReflecting the extent of excretion of the drug from the bile.

In sample group A and sample group B, C_t、AUC_0-t、AUC_0-∞CL as a dose-dependent plasma pharmacokinetic parameter_tot,p、V_ssAs a dose-independent plasma pharmacokinetic parameter, f_e-U、f_e-B、CL_R、CL_BPerforming sample T test as pharmacokinetic parameter reflecting excretion, calculating the pharmacokinetic parameter by SPSS19.0 software, and calculating when P is<At 0.05, a statistically significant difference is suggested, and the drug-induced matrix effect of key components in the Chinese patent medicine contained in the body fluid of the experimental mouse is determined.

The sample T test is calculated according to equation (8), where equation (8) is:

wherein the content of the first and second substances,

is the mean of two samples; sigma_x1 ²、σ_x2 ²The two recipes are different; gamma is the correlation coefficient of the correlation sample; n is the sample size. The equation (8) is calculated by using SPSS19.0 software, wherein the correlation coefficient γ of the correlation sample is calculated by fitting the SPSS19.0 software.