Rhizoma bletillae pharmacokinetic analysis and research method

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a rhizoma bletillae pharmacokinetic analysis and research method. According to the present invention, the ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology is adopted to establish the analysis method for simultaneously determining gastrodin (B1), alpha-isobutyl malic acid (B6), 4-(glucose oxygen)-cinnamic acid glucose oxygen benzyl ester (12), 1, 4-di [4-(glucose oxygen) benzyl]-2-isobutyl malate (B14) and 4-(glucose oxygen) benzyl-2-isobutyl malate (B17) in the mouse plasma; the method can be used for simultaneously and accurately measuring the blood concentration of each component in the effective part of rhizoma bletillae at each time point after single administration and multiple administration in a whole body heparinization bleeding model mouse body, and can be used for further revealing the time-dependent change rule of the five components in the bleeding model mouse body. The influence of the effective part of the rhizoma bletillae on the in-vivo process in a pathological state is further researched.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, in particular to a method for analyzing the pharmacokinetic analysis of Bletilla striata. Background Art

[0002] Bletilla striata is the dried tuber of Bletilla striata (Thunb.) Reichb.f., a plant of the orchid family. It is a commonly used medicinal material included in the Chinese Pharmacopoeia. It has the effects of reducing swelling, promoting tissue regeneration, astringing and stopping bleeding. It is often used for hemoptysis, traumatic bleeding, hematemesis, upper gastrointestinal bleeding and other diseases. The research team's previous studies found that the effective parts of Bletilla striata can stimulate the activation of rat platelets, enhance platelet aggregation, increase the generation of thrombin in the common coagulation pathway, and inhibit plasmin from converting fibrin into fibrin degradation products, thereby increasing blood viscosity and slowing blood flow to play a hemostatic effect. The pharmacokinetic behavior of drugs in the human body is closely related to the physiological state of the human body. Traditional Chinese medicine pharmacokinetic research is mostly based on healthy experimental animals, ignoring the fact that physiological and pathological changes in disease states may cause significant changes in related pharmacokinetic parameters. However, drugs are used to treat diseases, and patients are the final consumers of drugs. Whether the body is in a diseased state has different degrees of influence on the absorption, distribution, metabolism, and excretion of drugs, which is closely related to whether clinical medication is safe and effective. At present, the relevant research on Bletilla striata mainly focuses on the fields of pharmacology, clinical research, and quality standards, and its in vivo processes are only studied in normal bodies. There are no reports on the in vivo processes of the effective parts of Bletilla striata under pathological conditions.

[0003] Therefore, it is urgent to find an accurate, reliable and specific analysis method for the pharmacokinetic study of Bletilla striata, and to use this method to determine the blood concentrations of gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, and 4-(glucose oxygen) benzyl-2-isobutylmalate in the plasma of mice with systemic heparinized bleeding model after oral administration of the effective parts of Bletilla striata. This is to further reveal the time-dependent changes of the five components in the bleeding model mice and further explore the effects of the effective parts of Bletilla striata on the in vivo processes under pathological conditions. Summary of the invention

[0004] In order to solve the above technical problems existing in the prior art, the present invention provides a method for analyzing and studying the pharmacokinetic characteristics of Bletilla striata, which is as follows:

[0005] A method for analyzing and studying the pharmacokinetic properties of Bletilla striata can simultaneously determine the concentrations of gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, and 4-(glucose oxygen) benzyl-2-isobutylmalate in plasma, characterized in that the method is carried out using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology, and the specific conditions are as follows:

[0006] Chromatographic conditions: ACE UltraCore Super C18 column, specification: 2.1 mm × 100 mm, 2.5 μm) column; flow rate 0.20 mL min -1 ; Column temperature 45°C; Injector temperature 25°C; Injection volume 1 μL; Internal standard puerarin; Mobile phase 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B); Gradient elution program is as follows: 0-1 min, 10% A; 1-2 min, 10%-30% A; 2-6 min, 30%-40% A; 6-6.1 min, 40%-90% A; 6.1-8.1 min, 90% A; 8.1-8.2 min, 90%-10% A; 8.2-12 min, 10% A;

[0007] Mass spectrometry conditions: electrospray ionization (ESI) source, positive and negative modes scanning simultaneously, capillary ionization voltage: 3 kV, ion source temperature: 120 °C; spray gas and backflush gas: N 2 , desolventizing gas flow rate: 650L·hr -1 , desolvation temperature: 350°C, scanning mode: selected ion monitoring (MRM), mass spectrometry data acquisition and processing software: Analyst, MultiQuant workstation.

[0008] Furthermore, the monitoring ion mass spectrometry conditions for quantitative analysis of the five components of gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, 4-(glucose oxygen) benzyl-2-isobutyl malate and puerarin internal standard solution are as follows:

[0009]

[0010] Furthermore, in the liquid chromatography-mass spectrometry tandem technique, the test solution is obtained by the following treatment: 50 μL of plasma is taken and placed in a 1.5 mL plastic centrifuge tube, 50 μL of methanol is added, and 50 ng·mL -120 μL of puerarin internal standard solution was added with 20 μL of 1% formic acid and 400 μL of methanol, vortexed for 1 min, ultrasonicated for 10 min, centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was placed in a centrifuge tube and incubated at 37°C for 1 h. 2 The residue was dried and dissolved in 500 μL 50% methanol aqueous solution, vortexed for 1 min, and ultrasonicated for 10 min at 4 °C and 12000 r / min. -1 After centrifugation for 10 min, the supernatant was taken for HPLC-MS / MS analysis.

[0011] Furthermore, in the technology of liquid chromatography and mass spectrometry in series, the reference solution is a methanol solution of gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, and 4-(glucose oxygen) benzyl-2-isobutyl malate, and its specific concentration is as follows:

[0012] Concentrations of mixed standard solutions of five components including gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, and 4-(glucose oxygen) benzyl-2-isobutylmalate (μg·mL) -1 )

[0013]

[0014] Furthermore, the reference solution is prepared by the following method: accurately weigh reference substances gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, and 4-(glucose oxygen) benzyl-2-isobutyl malate, and dilute to 10 mL with methanol to obtain concentrations of 1.0, 1.1, 1.4, 1.1, 1.3, and 1.2 mg·mL, respectively. -1 Accurately measure the above 5 reference substance stock solutions respectively, and then dilute them step by step with methanol to obtain a mixed series of standard solutions.

[0015] Furthermore, the puerarin internal standard solution is a puerarin methanol solution with a concentration of 50 ng / mL.

[0016] Furthermore, the plasma is prepared by the following steps: selecting successfully modeled mice, 6 mice at each time point, and taking blood at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 8, 12, 24, 36, and 48 hours after oral administration of Bletilla striata extract (90.9 g / kg) to the single-dose group; taking blood at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 8, 12, 24, 36, and 48 hours after administration on the 4th day of the multiple-dose group; the blood was collected by puncturing the mouse's posterior orbital puncture to collect about 0.8 mL of blood, placing it in a centrifuge tube coated with sodium citrate, and centrifuging at 6000 r·min -1 The plasma was separated by centrifugation for 10 min and stored in a -80°C refrigerator.

[0017] Furthermore, the model mice were prepared by the following steps: 20 Kunming mice (half male and half female) were randomly divided into 2 groups, 10 mice in each group; divided into a blank group and a heparinized model group; the mice in the heparinized model group were injected with sodium heparin via the tail vein, 0.8 U / mouse; the blank group was injected with an equal amount of normal saline via the tail vein, about 0.8 mL of blood was collected by puncturing the mouse's posterior orbital puncture, and the blood was placed in a centrifuge tube coated with sodium citrate, and the blood was centrifuged at 6000 r·min -1 The mixture was centrifuged for 10 min to separate plasma, which was then used for the determination of TT, PL, TXB2, and Fbg.

[0018] Furthermore, the Bletilla striata extract is prepared by the following steps: weighing Bletilla striata medicinal material, refluxing and extracting it 3 times with 4 times the amount of 95% ethanol solution, 2h / time, filtering, combining the filtrate, concentrating to an extract, subjecting the extract to D101 macroporous adsorption resin column chromatography, eluting with water, and then eluting with 80% ethanol solution, collecting the eluate, concentrating under reduced pressure, and drying the residue in a microwave vacuum to obtain the Bletilla striata extract.

[0019] Furthermore, all raw data were calculated for pharmacokinetic parameters using WinNonlin 6.4 software, and the drug kinetics in mice were fitted with a non-compartmental model; SPSS 22.0 statistical software was used for data analysis, and the experimental results were expressed as X±S.

[0020] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0021] (1) The present invention adopts ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology to establish an analytical method for simultaneously determining gastrodin (B1), α-isobutylmalic acid (B6), 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester (12), 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate (B14), and 4-(glucose oxygen) benzyl-2-isobutyl malate (B17) in mouse plasma, and can simultaneously and accurately determine the blood drug concentrations of each component in the effective part of Bletilla striata at various time points after single and multiple administrations in mice with systemic heparinized bleeding model.

[0022] (2) The present invention uses an ACE UltraCore Super C18 (2.1 mm×100 mm, 2.5 μm) chromatographic column for ultra-high performance liquid chromatography (UPLC) analysis, and uses a gradient elution of 0.1% formic acid acetonitrile and 0.1% formic acid water, which has high separation, good peak shape and sensitivity.

[0023] (3) The present invention uses multiple reaction monitoring (MRM) to simultaneously scan the positive and negative ions of the five index components, and optimizes the parameters with the highest response. The method of the present invention can simultaneously determine the five index components and has the advantages of short detection time, high sensitivity, and strong specificity.

[0024] (4) The present invention uses puerarin as the internal standard, which is stable in nature, has a high response, and is completely separated from the five index components.

[0025] (5) The present invention uses methanol as the extraction solvent. Methanol has a better extraction effect than acetonitrile and methanol-acetonitrile (1:1) ethyl acetate extraction solvents, and has less interference from endogenous substances. The matrix effect and extraction recovery rate are better than other extraction solvents.

[0026] (6) This application finally obtained an accurate, reliable and highly specific analysis method for the pharmacokinetic study of Bletilla striata, and this method can be used to determine the blood drug concentrations of gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, and 4-(glucose oxygen) benzyl-2-isobutylmalate in the plasma of mice with systemic heparinized bleeding model after oral administration of the effective part of Bletilla striata, so as to further reveal the time-dependent changes of the five components in the mice with bleeding model, and further explore the influence of the effective part of Bletilla striata on the in vivo process under pathological conditions. And the specificity, precision, accuracy, accuracy, recovery rate, linear range and stability of this analysis method all meet the analysis requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1It is the chromatogram of blank plasma, blank plasma plus five index components including gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, 4-(glucose oxygen) benzyl-2-isobutylmalate and other six substances including internal standard, and plasma samples after administration to mice, among which A. blank plasma; B. blank plasma plus reference solution; C. measured sample.

[0028] Figure 2 This is the Ct drug-time curve of each component after mice orally administered Bletilla striata extract. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0030] Example:

[0031] Materials and methods

[0032] 1. Instruments and reagents

[0033] API 5500 / Q-TRAP high performance liquid chromatography-triple quadrupole tandem mass spectrometer; EL204 1 / 10,000 electronic balance (Mettler-Toledo Instrument Shanghai Co., Ltd.); Allegra 64R low-temperature high-speed centrifuge (Beckman Coulter, USA); CQ250A-TS ultrasonic cleaning machine (Shanghai Yuejin Medical Optical Instrument Factory); WP-UP-II-20 ultrapure water machine (Sichuan Wottel Technology Development Co., Ltd.); MTN-2800D nitrogen blowing concentration device (Tianjin Autes Instrument Co., Ltd.); XYN-15LP nitrogen blowing instrument (Shanghai Xiyou Analytical Instrument Co., Ltd.).

[0034] The medicinal material of Bletilla striata was picked from Zheng'an County, Guizhou Province (batch number: 20160510) and was identified by Associate Professor Long Qingde of the Department of Pharmacognosy and Medicinal Plants, School of Pharmacy, Guizhou Medical University as the dried tuber of Bletilla striata (Thunb.) Reichb.F., an orchid plant. Effective part of Bletilla striata (homemade, batch number: 2016718; content of each component: B1: 5.93%, B6: 2.06%, B12: 2.64%, B14: 26.17%, B17: 0.78%, B19: 0.64%, B23: 1.68%); sodium carboxymethyl cellulose (batch number 20151123, Chengdu Jinshan Chemical Reagent Co., Ltd.); sodium heparin injection (batch number 51506101, Jiangsu Wanbang Biochemical Pharmaceutical Co., Ltd.); 0.9% sodium chloride injection (batch number E118092502, Guizhou Kelun Pharmaceutical Co., Ltd.); sodium citrate (batch number: 616C0520, Beijing Solebow Technology Co., Ltd.); puerarin reference substance (batch number: 11 0752-201615, China Food and Drug Inspection Institute); Gastrodin (B1) reference substance (Batch No.: 110807-201608, China Food and Drug Inspection Institute); 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate (B14) reference substance (Batch No.: PS180413-03 Chengdu Pusi Biotechnology Co., Ltd.); 4-(glucose oxygen)-cinnamic acid glucose oxygen benzyl ester (12); 2-isobutylmalic acid (B6), 4-(glucose oxygen) benzyl-2-isobutylmalic acid (B17) reference substances were isolated from Bletilla striata in the laboratory and identified by 1H, 13C-NMR, MS, UV and IR spectra. The chemical structures are shown in Figure 1 , TLC purity check showed a single spot, and the peak area normalized content was greater than 95% when detected by UPLC-PDA at multiple wavelengths;

[0035] 2 Animals

[0036] Clean-grade KM mice, half male and half female, weighing 18-22 g; provided by the Guizhou Medical University Animal Room, license number: SCXK (Guizhou) 2018-0001, approved by the Guizhou Medical University Animal Experiment Ethics Committee (approval number 1603125). After the mice were introduced into the laboratory, they were divided into 8 mice per cage, and female mice were raised separately from male mice. The animal room had sufficient light, good air conditioning and ventilation systems, and the temperature was controlled at 18-25°C and the relative humidity was 50%-60%.

[0037] 3. Solution preparation

[0038] 3.1 Preparation of standard solution

[0039] Accurately weigh appropriate amounts of reference substances B1, B6, B12, B14, and B17, and dilute to 10 mL with methanol to obtain concentrations of 1.0, 1.1, 1.4, 1.1, 1.3, and 1.2 mg mL, respectively. -1 Precisely measure the appropriate amount of the above 5 reference substance stock solutions, dilute them step by step with methanol, and make up to the volume to obtain a mixed series of standard solutions, see the table below. Store in a refrigerator (-20℃). Prepare a puerarin internal standard solution with a mass concentration of 50ng / mL in the same way and store in a refrigerator (-20℃).

[0040] Table 1 Concentrations of mixed standard solutions of five components including B1 (μg·mL -1 )

[0041]

[0042]

[0043] 3.2 Preparation of Heparin Sodium Injection

[0044] Take an appropriate amount of heparin sodium injection and dilute it with normal saline to make 4U·mL -1 Active concentration, ready for use.

[0045] 4. Preparation of effective parts of Bletilla striata

[0046] Weigh an appropriate amount of Bletilla striata medicinal material, reflux extract with 4 times the amount of 95% ethanol solution for 3 times, 2h / time, filter, combine the filtrate, concentrate to an extract, apply the extract to D101 macroporous adsorption resin column chromatography, elute with water, and then elute with 80% ethanol solution, collect the eluate, concentrate under reduced pressure, and microwave vacuum dry the residue to obtain the effective part of Bletilla striata, with an extraction rate of 6.82%.

[0047] 5UPLC-MS / MS analysis

[0048] 5.1 Chromatographic conditions

[0049] The chromatographic column was an ACE UltraCore Super C18 (2.1 mm × 100 mm, 2.5 μm) column; the flow rate was 0.20 mL min-1; the column temperature was 45 °C; the temperature of the injector was 25 °C; the injection volume was 1 μL; the internal standard was puerarin; the mobile phase was 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B); the gradient elution was (0-1 min, 10% A; 1-2 min, 10%-30% A; 2-6 min, 30%-40% A; 6-6.1 min, 40%-90% A; 6.1-8.1 min, 90% A; 8.1-8.2 min, 90%-10% A; 8.2-12 min, 10% A).

[0050] 5.2 Mass spectrometry conditions

[0051] Electrospray ionization (ESI) was used, with positive and negative modes scanning simultaneously, capillary ionization voltage: 3 kV, ion source temperature: 120 °C; spray gas and backflush gas: N 2 , desolventizing gas flow rate: 650L·hr -1 , desolvation temperature: 350℃, scanning mode: selected ion monitoring (MRM), mass spectrometry data acquisition and processing software: Analyst, MultiQuant workstation. The monitoring ions of 5 components including B1 and internal standard used for quantitative analysis are shown in the table below.

[0052] Table 2 Mass spectrometry conditions

[0053]

[0054] 6. Processing of plasma samples

[0055] Take 50 μL of mouse plasma and place it in a 1.5 mL plastic centrifuge tube. Add 50 μL of methanol and 50 ng mL -1 20 μL of internal standard solution was added with 20 μL of 1% formic acid and 400 μL of methanol, vortexed for 1 min, ultrasonicated for 10 min, centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was placed in a centrifuge tube and incubated at 37°C for 1 h. 2 The residue was dried and dissolved in 500 μL 50% methanol aqueous solution, vortexed for 1 min, and ultrasonicated for 10 min at 4 °C and 12000 r / min. -1 After centrifugation for 10 min, the supernatant was taken for HPLC-MS / MS analysis.

[0056] 7. Preparation of heparinized bleeding model mice

[0057] Twenty Kunming mice (half male and half female) were randomly divided into two groups, 10 in each group. They were divided into a blank group and a heparinized model group. The mice in the heparinized model group were injected with sodium heparin, 0.8 U / mouse, through the tail vein; the blank group was injected with an equal amount of normal saline through the tail vein, and about 0.8 mL of blood was collected by puncturing the mouse's posterior orbital puncture, placed in a centrifuge tube coated with sodium citrate, and centrifuged at 6000 r·min -1 The mixture was centrifuged for 10 min to separate plasma, which was then used for the determination of TT, PL, TXB2, and Fbg.

[0058] 8 Methodological Investigation

[0059] 8.1 Specificity Investigation

[0060] Take 50 μL of blank mouse plasma, and operate according to the plasma sample processing method under "6" except for not adding puerarin internal standard, to obtain a blank sample chromatogram; add a certain concentration of standard solution and internal standard solution to the blank plasma, operate according to the method, and obtain the corresponding chromatogram; take the plasma of mice after administration, and operate according to the method to obtain the corresponding chromatogram.

[0061] 8.2 Standard curve and linear range investigation

[0062] Take 50 μL of blank mouse plasma, add 50 μL of mixed standard solution, and perform sampling analysis according to the plasma sample processing method under "6". Use the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard (A / Ai) as the ordinate Y, and the concentration of each substance (C) as the abscissa X for linear regression, with a weight coefficient of 1 / X, to obtain the linear regression equations of B1, B6, B12, B14, and B17. The lowest limit of quantification (LLOQ) is defined as S / N ≥ 10, and the lowest detection (LLOD) is defined as S / N ≥ 3.

[0063] 8.3 Precision and Accuracy Examination

[0064] Prepare three quality control (QC) samples of five components of mouse plasma at low, medium and high concentrations according to item "8.2", and operate according to the plasma sample processing method under item "6". Perform 6 sample analyses for each concentration, inject samples continuously within the day, and measure continuously for 3 days, and perform the analysis simultaneously with the standard curve. Calculate the concentration of the QC sample according to the standard curve of the day, compare it with the prepared concentration, and perform variance analysis on the results of the QC sample to obtain the precision (standard deviation of the QC sample measurement value) and accuracy (relative error between the QC sample measurement mean and the true value) of this method. 8.4 Investigation of extraction recovery and matrix effect

[0065] Take 50 μL of mouse blank plasma, prepare three quality control samples (QC) of low, medium and high concentrations according to item "8.2", and perform 6 replicates for each concentration, and perform the operation according to item "6" (sample A); take another 50 μL of blank plasma, except for not adding the mixed standard solution, perform the operation according to item "6", add the corresponding low, medium and high concentration mixed standard solution and internal standard to the obtained supernatant, blow dry, and dissolve the residue with 500 μL 50% methanol aqueous solution (sample B); take another low, medium and high concentration mixed standard solution and internal standard, blow dry, and dissolve the residue with 500 μL 50% methanol aqueous solution (sample C). The extraction recovery rate is calculated by the ratio of the chromatographic peak area of ​​sample B to that of sample A, and the matrix effect is calculated by the ratio of the chromatographic peak area of ​​sample B to that of sample C.

[0066] 8.5 Stability Study

[0067] According to item "8.2", three plasma quality control samples (QC) of low, medium and high concentrations of five components were prepared respectively. After the samples were processed, they were put into the autosampler and injected at 6 hours to investigate the stability of five components such as B1 in the treated plasma samples under the conditions of the autosampler. Six samples were analyzed for each concentration. Three plasma samples (QC) of low, medium and high concentrations were prepared in the same way, frozen and thawed three times, and injected after processing to determine the concentration, and investigate the stability of five components such as B1 in plasma under repeated freezing and thawing conditions.

[0068] 9 Pharmacokinetic studies

[0069] The model mice with successful modeling were selected, 6 mice at each time point. After the single-dose group was gavaged with Bletilla striata extract (90.9g / kg), blood was collected at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 8, 12, 24, 36, and 48h. The mice in the multiple-dose group were gavaged with the effective part of Bletilla striata (dose 90.9g / kg) once a day for 4 consecutive days. After the administration on the 4th day, blood was collected at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 8, 12, 24, 36, and 48h. The blood collection method was to collect about 0.8mL of blood through puncture of the mouse's posterior orbit, place it in a centrifuge tube coated with sodium citrate, centrifuge it at 6000r·min-1 for 10min, separate the plasma, and store it in a -80℃ refrigerator.

[0070] 10 Pharmacokinetic Data Processing

[0071] WinNonlin 6.4 software was used to calculate the pharmacokinetic parameters, and the non-compartmental model was fitted for the kinetic process of the drug in mice. SPSS22.0 statistical software was used for data analysis, and the experimental results were used to express.

[0072] result

[0073] 1 Determination of TT, PL, TXB2 and Fbg in hemorrhagic model mice

[0074] After sodium heparin injection was injected into the tail vein of mice, the thrombin time (TT) of mice was significantly increased compared with the blank group (P<0.01); the levels of plasmin (PL), thromboxane B2 (TXB2) and fibrinogen (Fbg) in mice were reduced, indicating that the mouse bleeding model was successfully established.

[0075] Table 3 Effects of sodium heparin injection on TT, PL and TXB in mice 2 , the influence of Fbg (mean±SD, n=10)

[0076]

[0077] Note: **P<0.01 vs blank group.

[0078] 2 Validation of UPLC-MS / MS analysis method

[0079] 2.1 Exclusivity

[0080] Under the experimental conditions, the monitoring ion reaction MS / MS of 5 index components and internal standard puerarin of B1, B6, B12, B14, B17 were m / z 304.3→107.2; m / z 189.0→129.0; m / z 593.2→431.1; m / z 725.3→457.3; m / z 457.1→127.0; m / z 417.1→267.1; the chromatograms of blank plasma, blank plasma plus 5 index components and internal standard and plasma samples after administration of mice are shown in Figure 1 The results showed that the components were well separated and the endogenous substances in plasma did not interfere with the determination of the components to be tested.

[0081] 2.2 Standard curve and linear range

[0082] The five components in mouse plasma, including B1, had good linear relationships within their linear ranges, and the correlation coefficients (r) of the standard curves of each component were all greater than 0.99. The limits of detection (LLOQ) and limits of quantification (LLOD) are shown in the table below.

[0083] Table 4 Regression curve equations of B1 and other five components

[0084]

[0085] 2.3 Precision and Accuracy

[0086] The intra-day and inter-day precision RSDs (%) of B1 and other five components were all less than 15%, and the accuracy range was 83.44-102.93%, indicating that the method was accurate, reliable, and reproducible, and met the requirements of biological sample analysis methods. The results are shown in the table below.

[0087] Table 5 Accuracy, intra-day and inter-day precision of B1 and other five components in rat plasma ( n=6,3days)

[0088]

[0089] 2.4 Extraction recovery and matrix effect

[0090] The extraction recovery and matrix effect of the five index components at low, medium and high concentrations were in the range of 83.83-102.30% and 85.53-103.30, respectively. The experimental results showed that the extraction recovery was good and there was no obvious matrix effect, which met the requirements of the biological sample analysis method. The results are shown in the table below.

[0091] Table 6 Extraction recovery and matrix effect of B1 and other five components in rat plasma ( n=6)

[0092]

[0093]

[0094] 2.5 Sample stability

[0095] The five components of plasma samples were relatively stable within 6 hours after being processed and placed in the autosampler and after three freeze-thaw cycles, meeting the requirements of the biological sample analysis method. The results are shown in the table below.

[0096] Table 7 Stability of B1 and other five components in rat plasma ( n=6)

[0097]

[0098] 2.6 Pharmacokinetic study

[0099] The established UPLC-MS / MS analysis method was used to determine the blood drug concentration at various time points after oral administration of the effective part of Bletilla striata in mice. The average blood drug concentration-time curve is shown in Figure 2 WinNonlin6.4 software was used to calculate the pharmacokinetic parameters (see the table below), and the non-compartmental model was fitted for the drug's kinetic process in mice.

[0100] Table 8 Main pharmacokinetic parameters of B1 and other five components ( n=6)

[0101]

[0102]

[0103] Note: **P<0.01vs single-dose group.

[0104] 3. Discussion

[0105] In this study, multiple reaction monitoring (MRM) was used to scan the positive and negative ions of the five index components simultaneously, and the parameters with the highest response were selected; puerarin was selected as the internal standard, which is stable in nature, has a high response, and is completely separated from the five index components; at the same time, in the treatment of plasma samples, it was found that methanol has a better extraction effect than acetonitrile, methanol-acetonitrile (1:1) ethyl acetate extraction solvent, and has less interference from endogenous substances, and the matrix effect and extraction recovery rate are better than other extraction solvents. The results show that the specificity, precision, accuracy, accuracy, recovery rate, linear range and stability of this analytical method meet the analytical requirements.

[0106] Pharmacokinetic studies have shown that the contents of B14 and B1 in the effective parts of Bletilla striata are 26.17% and 5.93%, respectively. However, in the single-dose group, the AUCs of B14 and B1 were 57.12±6.44 and 625.44±48.85 h*mg / L, respectively; in the multiple-dose group, the AUCs of B14 and B1 were 213.18±27.53 and 934.50±97.45 h*mg / L, respectively. It can be seen that the AUC of B1 is much greater than that of B14. In addition, multiple peaks appeared in the drug-time curves of B1, B6 and B17. According to studies, B14 can be converted into B1, B6 and B17 in the body through ester bond hydrolysis, and the ester bond hydrolysis of B17 can be converted into B1 and B6. Therefore, although the content of B1 is much less than that of B14, B14 and B17 are continuously converted into B1 in the body, so that B1 accumulates continuously and has a larger AUC. One of the reasons for the multi-peak phenomenon in the drug-time curves of B1, B6 and B17 may be that B14 is continuously converted in the body. After a single dose, the five representative ingredients were poorly absorbed in systemic heparinized mice. The AUC and Cmax in the multiple-dose group were significantly higher than those in the single-dose group. Except for B1, the clearance rates of the other four active ingredients were reduced, indicating that the absorption of the multiple-dose group in the systemic heparinized bleeding model mice was better than that of the single-dose group, and the bioavailability was greatly increased, which is conducive to its rapid achievement of effective therapeutic concentrations in the diseased body, thereby exerting its pharmacological effects, and its mechanism still needs further pharmacological and pharmacokinetic-pharmacodynamic (PK-PD) studies.

[0107] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for analyzing the pharmacokinetic properties of Bletilla striata, which can simultaneously determine the concentrations of gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, and 4-(glucose oxygen) benzyl-2-isobutylmalate in plasma, characterized in that: Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology was used, and the specific conditions were as follows: Chromatographic conditions: ACE UltraCore Super C18 column, specification: 2.1 mm × 100 mm, 2.5 μm column; flow rate 0.20 mL min -1 ; Column temperature 45°C; Injector temperature 25°C; Injection volume 1 μL; Internal standard puerarin; Mobile phase 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B); Gradient elution program is as follows: 0-1 min, 10% A; 1-2 min, 10%-30% A; 2-6 min, 30%-40% A; 6-6.1 min, 40%-90% A; 6.1-8.1 min, 90% A; 8.1~8.2min, 90%~10%A; 8.2~12min, 10%A; Mass spectrometry conditions: electrospray ionization source, positive and negative modes scanning simultaneously, capillary ionization voltage: 3 kV, ion source temperature: 120 °C; spray gas and backflush gas: N2, desolvation gas flow rate: 650 L·hr -1 , desolvation temperature: 350°C, scanning mode: selected ion monitoring, mass spectrometry data acquisition and processing software: Analyst, MultiQuant workstation.

2. The pharmacokinetic analysis and research method of Bletilla striata according to claim 1, characterized in that: The monitoring ion mass spectrometry conditions for the quantitative analysis of the five components of gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, 4-(glucose oxygen) benzyl-2-isobutyl malate and puerarin internal standard solution are as follows:

3. The pharmacokinetic analysis and research method of Bletilla striata according to claim 1, characterized in that: The liquid chromatography-mass spectrometry technology is used in series, wherein the test solution is obtained by the following treatment: 50 μL of plasma is taken and placed in a 1.5 mL plastic centrifuge tube, 50 μL of methanol is added, and 50 ng·mL -1 20 μL of puerarin internal standard solution was added with 20 μL of 1% formic acid and 400 μL of methanol, vortexed for 1 min, ultrasonicated for 10 min, centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was placed in a centrifuge tube, dried with N2 at 37°C, the residue was dissolved with 500 μL of 50% methanol aqueous solution, vortexed for 1 min, ultrasonicated for 10 min, and centrifuged at 4°C and 12000 r·min -1 After centrifugation for 10 min, the supernatant was taken for HPLC-MS / MS analysis.

4. The pharmacokinetic analysis and research method of Bletilla striata according to claim 1, characterized in that: The liquid chromatography-mass spectrometry tandem technology, wherein the reference solution is a methanol solution of gastrodin, α-isobutyl malic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, 4-(glucose oxygen) benzyl-2-isobutyl malate, and its specific concentration is as follows: Concentrations of mixed standard solutions of five components including gastrodin, α-isobutylmalic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, and 4-(glucose oxygen) benzyl-2-isobutylmalate (μg·mL) -1 ) 5. The pharmacokinetic analysis and research method of Bletilla striata according to claim 1, characterized in that: The reference solution is prepared by the following method: accurately weigh reference substances gastrodin, α-isobutyl malic acid, 4-(glucose oxygen)-cinnamic acid glucose oxybenzyl ester, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, and 4-(glucose oxygen) benzyl-2-isobutyl malate, and dilute to 10 mL with methanol to obtain concentrations of 1.0, 1.1, 1.4, 1.1, 1.3, and 1.2 mg·mL, respectively. -1 Accurately measure the above 5 reference substance stock solutions respectively, and then dilute them step by step with methanol to obtain a mixed series of standard solutions.

6. The pharmacokinetic analysis and research method of Bletilla striata according to claim 2, characterized in that: The puerarin internal standard solution is a puerarin methanol solution with a concentration of 50 ng / mL.

7. The pharmacokinetic analysis and research method of Bletilla striata according to claim 2, characterized in that: The plasma is prepared by the following steps: selecting successfully modeled mice, 6 mice at each time point, and taking blood at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 8, 12, 24, 36, and 48 hours after oral administration of Bletilla striata extract (90.9 g / kg) to the single-dose group; taking blood at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 8, 12, 24, 36, and 48 hours after oral administration to the mice in the multiple-dose group; taking blood at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 8, 12, 24, 36, and 48 hours after administration on the 4th day; the blood collection method is to collect about 0.8 mL of blood by puncturing the mouse's posterior orbit, put it in a centrifuge tube coated with sodium citrate, and centrifuge it at 6000 r·min -1 The plasma was separated by centrifugation for 10 min and stored in a -80°C refrigerator.

8. The pharmacokinetic analysis and research method of Bletilla striata according to claim 7, characterized in that: The model mice are prepared by the following steps: 20 Kunming mice (half male and half female) are randomly divided into 2 groups, 10 mice in each group; divided into a blank group and a heparinized model group; the mice in the heparinized model group are injected with sodium heparin via the tail vein, 0.8 U / mouse; the blank group is injected with an equal amount of physiological saline via the tail vein, about 0.8 mL of blood is collected by puncturing the mouse's posterior orbit, and the blood is placed in a centrifuge tube coated with sodium citrate, and the blood is centrifuged at 6000 r·min -1 The mixture was centrifuged for 10 min to separate plasma, which was then used for the determination of TT, PL, TXB2, and Fbg.

9. The pharmacokinetic analysis and research method of Bletilla striata according to claim 7, characterized in that: The Bletilla striata extract is prepared by the following steps: weighing Bletilla striata medicinal material, refluxing and extracting it three times with 4 times the amount of 95% ethanol solution, 2 hours each time, filtering, combining the filtrate, concentrating to an extract, subjecting the extract to D101 macroporous adsorption resin column chromatography, eluting with water, and then eluting with 80% ethanol solution, collecting the eluate, concentrating under reduced pressure, and drying the residue in a microwave vacuum to obtain the Bletilla striata extract.

10. The pharmacokinetic analysis and research method of Bletilla striata according to claim 1, characterized in that: All raw data were calculated using WinNonlin 6.4 software to calculate pharmacokinetic parameters, and the drug kinetics in mice were fitted with a non-compartmental model; SPSS22.0 statistical software was used for data analysis, and the experimental results were expressed as X±S.

Images